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UnAIDed Class Switching in Activated B-Cells Reveals Intrinsic Features of a Self-Cleaving IgH Locus

ABSTRACT: Activation-induced deaminase (AID) is the major actor of immunoglobulin (Ig) gene diversification in germinal center B-cells. From its first description, it was considered as mandatory for class switch recombination (CSR), and this discovery initiated a long quest for all of the AID-interacting factors controlling its activity. The mechanisms focusing AID-mediated DNA lesions to given target sequences remain incompletely understood with regards the detailed characterization of optimal substrates in which cytidine deamination will lead to double strand breaks (DSBs) and chromosomal cleavage. In an effort to reconsider whether such CSR breaks absolutely require AID, we herein provide evidence, based on deep-sequencing approaches, showing that this dogma is not absolute in both human and mouse B lymphocytes. In activated B-cells from either AID-deficient mice or human AID-deficient patients, we report an intrinsic ability of the IgH locus to undergo “on-target” cleavage and subsequent synapsis of broken regions in conditions able to yield low-level CSR. DNA breaks occur in such conditions within the same repetitive S regions usually targeted by AID, but their repair follows a specific pathway with increased usage of microhomology-mediated repair. These data further demonstrate the role of AID machinery as not initiating de novo chromosomal cleavage but rather catalyzing a process which spontaneously initiates at low levels in an appropriately conformed IgH locus.

SUBMITTER: Dalloul I

PROVIDER: S-EPMC8581400 | biostudies-literature |

REPOSITORIES: biostudies-literature

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UnAIDed Class-Switching in activated B-cells reveals intrinsic features of a self-cleaving IgH locus

Project description:Activation-induced deaminase (AID) is the major actor of immunoglobulin (Ig) genes diversification in germinal center B cells. From its first description, it was considered as mandatory for class switch recombination (CSR) and this discovery initiated a long quest for all of the AID-interacting factors controlling its activity. The mechanisms focusing AID-mediated DNA lesions to given target sequences remain incompletely understood, with regards to the detailed characterization of optimal substrates in which cytidine deamination will lead to double strand breaks (DSBs) and chromosomal cleavage. In an effort to reconsider whether such CSR breaks absolutely require AID, we herein provide evidence refuting this dogma in both human and mouse B lymphocytes, based on deep-sequencing approaches. In activated B-cells from either AID-deficient mice or human AID-deficient patients, we report an intrinsic ability of the IgH locus to undergo “on-target” cleavage and subsequent synapsis of broken regions in conditions able to yield low-level CSR. DNA breaks occur in such conditions within the same repetitive S regions usually targeted by AID, but their repair follows a specific pathway with increased usage of microhomology-mediated repair. These data further demonstrate the role of AID machinery as not initiating de novo chromosomal cleavage, but rather catalyzing a process which spontaneously initiates at low levels in an appropriately conformed IgH locus.

2021-10-26 | GSE183034 | GEO

UnAIDed Class-Switching in activated B-cells reveals intrinsic features of a self-cleaving IgH locus

Project description:UnAIDed Class-Switching in activated B-cells reveals intrinsic features of a self-cleaving IgH locus

| PRJNA758901 | ENA

53BP1 Contributes to Igh Locus Chromatin Topology during Class Switch Recombination.

Project description:In B lymphocytes, Ig class switch recombination (CSR) is induced by activation-induced cytidine deaminase, which initiates a cascade of events leading to DNA double-strand break formation in switch (S) regions. Resolution of DNA double-strand breaks proceeds through formation of S-S synaptic complexes. S-S synapsis is mediated by a chromatin loop that spans the C region domain of the Igh locus. S-S junctions are joined via a nonhomologous end joining DNA repair process. CSR occurs via an intrachromosomal looping out and deletion mechanism that is 53BP1 dependent. However, the mechanism by which 53BP1 facilitates deletional CSR and inhibits inversional switching events remains unknown. We report a novel architectural role for 53BP1 in Igh chromatin looping in mouse B cells. Long-range interactions between the E? and 3'E? enhancers are significantly diminished in the absence of 53BP1. In contrast, germline transcript promoter:3'E? looping interactions are unaffected by 53BP1 deficiency. Furthermore, 53BP1 chromatin occupancy at sites in the Igh locus is B cell specific, is correlated with histone H4 lysine 20 marks, and is subject to chromatin spreading. Thus, 53BP1 is required for three-dimensional organization of the Igh locus and provides a plausible explanation for the link with 53BP1 enforcement of deletional CSR.

| S-EPMC5695034 | biostudies-literature

Physiological role of the 3'IgH CBEs super-anchor in antibody class switching.

Project description:IgH class switch recombination (CSR) replaces Cμ constant region (CH) exons with one of six downstream CHs by joining transcription-targeted double-strand breaks (DSBs) in the Cμ switch (S) region to DSBs in a downstream S region. Chromatin loop extrusion underlies fundamental CSR mechanisms including 3'IgH regulatory region (3'IgHRR)-mediated S region transcription, CSR center formation, and deletional CSR joining. There are 10 consecutive CTCF-binding elements (CBEs) downstream of the 3'IgHRR, termed the "3'IgH CBEs." Prior studies showed that deletion of eight 3'IgH CBEs did not detectably affect CSR. Here, we report that deletion of all 3'IgH CBEs impacts, to varying degrees, germline transcription and CSR of upstream S regions, except that of Sγ1. Moreover, deletion of all 3'IgH CBEs rendered the 6-kb region just downstream highly transcribed and caused sequences within to be aligned with Sμ, broken, and joined to form aberrant CSR rearrangements. These findings implicate the 3'IgH CBEs as critical insulators for focusing loop extrusion-mediated 3'IgHRR transcriptional and CSR activities on upstream CH locus targets.

| S-EPMC7826415 | biostudies-literature

SHLD2 promotes class switch recombination by preventing inactivating deletions within the Igh locus.

Project description:The newly identified shieldin complex, composed of SHLD1, SHLD2, SHLD3, and REV7, lies downstream of 53BP1 and acts to inhibit DNA resection and promote NHEJ. Here, we show that Shld2-/- mice have defective class switch recombination (CSR) and that loss of SHLD2 can suppress the embryonic lethality of a Brca1Δ11 mutation, highlighting its role as a key effector of 53BP1. Lymphocyte development and RAG1/2-mediated recombination were unaffected by SHLD2 deficiency. Interestingly, a significant fraction of Shld2-/- primary B-cells and 53BP1- and shieldin-deficient CH12F3-2 B-cells permanently lose expression of immunoglobulin upon induction of CSR; this population of Ig-negative cells is also seen in other NHEJ-deficient cells and to a much lesser extent in WT cells. This loss of Ig is due to recombination coupled with overactive resection and loss of coding exons in the downstream acceptor constant region. Collectively, these data show that SHLD2 is the key effector of 53BP1 and critical for CSR in vivo by suppressing large deletions within the Igh locus.

| S-EPMC7403657 | biostudies-literature

Crystal structure of Pistol, a class of self-cleaving ribozyme.

Project description:Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile by deprotonating the 2'-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5' leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.

| S-EPMC5293083 | biostudies-literature

A widespread self-cleaving ribozyme class is revealed by bioinformatics.

Project description:Ribozymes are noncoding RNAs that promote chemical transformations with rate enhancements approaching those of protein enzymes. Although ribozymes are likely to have been abundant during the RNA world era, only ten classes are known to exist among contemporary organisms. We report the discovery and analysis of an additional self-cleaving ribozyme class, called twister, which is present in many species of bacteria and eukarya. Nearly 2,700 twister ribozymes were identified that conform to a secondary structure consensus that is small yet complex, with three stems conjoined by internal and terminal loops. Two pseudoknots provide tertiary structure contacts that are critical for catalytic activity. The twister ribozyme motif provides another example of a natural RNA catalyst and calls attention to the potentially varied biological roles of this and other classes of widely distributed self-cleaving RNAs.

| S-EPMC3867598 | biostudies-literature

Intrinsic transcriptional heterogeneity in B cells controls early class switching to IgE

Project description:'Non-coding transcripts originating upstream of the immunoglobulin constant region (I-transcripts) are required to direct activation-induced deaminase to initiate class switching in B cells. Differential regulation of I and I1 transcription in response to interleukin-4 (IL-4), hence class switching to IgE and IgG1, is not fully understood. Here we combine novel mouse reporters and single-cell RNA-seq to reveal the heterogeneity in IL-4 induced I-transcription. Transgenic mice were generated with one allele labelled with an eGFP fluorescent reporter gene in the first exon of the gamma-1 non-coding RNA and one allele with a tdTom fluorescent reporter gene in the first exon of the epsilon non-coding RNA. Ex vivo splenic B cells from these mice were stimulated with IL-4, lipopolysaccharide and B cell activating factor. After 24 hours culture, cell were sorted according to their fluorescence and used for single-cell mRNA-sequencing. Two independent experiments were performed (labelled as ''batch 1'' and ''batch 2'').'

2016-12-08 | E-MTAB-4825 | biostudies-arrayexpress

A PTIP-PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex.

Project description:Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities.

| S-EPMC4719306 | biostudies-literature

Intrinsic transcriptional heterogeneity in B cells controls early class switching to IgE.

Project description:Noncoding transcripts originating upstream of the immunoglobulin constant region (I transcripts) are required to direct activation-induced deaminase to initiate class switching in B cells. Differential regulation of Iε and Iγ1 transcription in response to interleukin 4 (IL-4), hence class switching to IgE and IgG1, is not fully understood. In this study, we combine novel mouse reporters and single-cell RNA sequencing to reveal the heterogeneity in IL-4-induced I transcription. We identify an early population of cells expressing Iε but not Iγ1 and demonstrate that early Iε transcription leads to switching to IgE and occurs at lower activation levels than Iγ1. Our results reveal how probabilistic transcription with a lower activation threshold for Iε directs the early choice of IgE versus IgG1, a key physiological response against parasitic infestations and a mediator of allergy and asthma.

| S-EPMC5206502 | biostudies-literature

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