Faculty Opinions recommendation of Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination.

Mature B cells express B cell antigen receptor (BCR), toll-like receptors (TLR) and TNF family receptors including CD40 and B-cell activating factor receptor (BAFFR). These receptors transduce cellular signals to govern the physiological and pathological processes in B cells including B cell development and differentiation, survival, proliferation, and antibody-mediated immune responses as well as autoimmune diseases and B cell lymphomagenesis. Effective antibody-mediated immune responses require class switch recombination (CSR), a somatic DNA recombination event occurring at the immunoglobulin heavy chain (Igh) gene locus. Mature B cells initially express IgM as their BCR, and CSR enables the B cells to switch from expressing IgM to expressing different classes of antibodies including IgG, IgA or IgE that exhibit distinct effector functions. Here, we briefly review recent findings about how the signaling crosstalk of the BCR with TLRs, CD40 and BAFFR regulates CSR, antibody-mediate immune responses, and B cell anergy.

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Non-coding Class Switch Recombination-related transcription in human normal and pathological immune responses

10.1101/384172 ◽

2018 ◽

Author(s):

Helena Kuri-Magaña ◽

Leonardo Collado-Torres ◽

Andrew E. Jaffe ◽

Humberto Valdovinos-Torres ◽

Marbella Ovilla-Muñoz ◽

...

Keyword(s):

B Cells ◽

Immune Responses ◽

Class Switch Recombination ◽

Rna Seq ◽

Ileal Mucosa ◽

Exon Expression ◽

Class Switch ◽

Cytokine Milieu ◽

Antibody Class ◽

Cancer Tissues

AbstractBackgroundAntibody class switch recombination (CSR) to IgG, IgA or IgE is a hallmark of adaptive immunity, allowing antibody function diversification beyond IgM. CSR involves a deletion of the IgM/IgD constant region genes placing a new acceptor Constant (CH) gene, downstream of the VDJH exon. CSR depends on non-coding (CSRnc) transcription of donor Iμ and acceptor IH exons, located 5’ upstream of each CH coding gene. Although our knowledge of the role of CSRnc transcription has advanced greatly, its extension and importance in healthy and diseased humans is scarce.MethodsWe analyzed CSRnc transcription in 70,603 publicly available RNA-seq samples, including GTEx, TCGA and the Sequence Read Archive (SRA) using recount2, an online resource consisting of normalized RNA-seq gene and exon counts, as well as coverage BigWig files that can be programmatically accessed through R. CSRnc transcription was validated with a qRT-PCR assay for Iμ, Iγ3 and Iγ1 in humans in response to vaccination.ResultsWe mapped IH transcription for the human IgH locus, including the less understood IGHD gene. CSRnc transcription was restricted to B cells and is widely distributed in normal adult tissues, but predominant in blood, spleen, MALT-containing tissues, visceral adipose tissue and some so-called “immune privileged” tissues. However, significant Iγ4 expression was found even in non-lymphoid fetal tissues. CSRnc expression in cancer tissues mimicked the expression of their normal counterparts, with notable pattern changes in some common cancer subsets. CSRnc transcription in tumors appears to result from tumor infiltration by B cells, since CSRnc transcription was not detected in corresponding tumor-derived immortal cell lines. Additionally, significantly increased I5 transcription in ileal mucosa in Crohn’s disease with ulceration was found.ConclusionsCSRnc transcription occurs in multiple anatomical locations beyond classical secondary lymphoid organs, representing a potentially useful marker of effector B cell responses in normal and pathological immune responses. The pattern of IH exon expression may reveal clues of the local immune response (i.e. cytokine milieu) in health and disease. This is a great example of how the public recount2 data can be used to further our understanding of transcription, including regions outside the known transcriptome.

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Mlh1 Can Function in Antibody Class Switch Recombination Independently of Msh2

Journal of Experimental Medicine ◽

10.1084/jem.20022190 ◽

2003 ◽

Vol 197 (10) ◽

pp. 1377-1383 ◽

Cited By ~ 46

Author(s):

Carol E. Schrader ◽

Joycelyn Vardo ◽

Janet Stavnezer

Keyword(s):

B Cells ◽

Somatic Hypermutation ◽

Class Switch Recombination ◽

Variable Region ◽

Class Switch ◽

Switch Regions ◽

Substitution Mutations ◽

Antibody Class ◽

Variable Region Genes ◽

Mmr Proteins

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 Sμ-Sγ3 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.

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Sγ3 switch sequences function in place of endogenous Sγ1 to mediate antibody class switching

Journal of Experimental Medicine ◽

10.1084/jem.20080451 ◽

2008 ◽

Vol 205 (7) ◽

pp. 1567-1572 ◽

Cited By ~ 11

Author(s):

Ali A. Zarrin ◽

Peter H. Goff ◽

Kate Senger ◽

Frederick W. Alt

Keyword(s):

B Cells ◽

Heavy Chain ◽

Recombination Event ◽

Class Switch Recombination ◽

Constant Region ◽

Wild Type ◽

Class Switch ◽

Specific Factors ◽

Antibody Class

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) replaces the initially expressed IgH Cμ exons with a set of downstream IgH constant region (CH) exons. Individual sets of CH exons are flanked upstream by long (1–10-kb) repetitive switch (S) regions, with CSR involving a deletional recombination event between the donor Sμ region and a downstream S region. Targeting CSR to specific S regions might be mediated by S region–specific factors. To test the role of endogenous S region sequences in targeting specific CSR events, we generated mutant B cells in which the endogenous 10-kb Sγ1 region was replaced with wild-type (WT) or synthetic 2-kb Sγ3 sequences or a synthetic 2-kb Sγ1 sequence. We found that both the inserted endogenous and synthetic Sγ3 sequences functioned similarly to a size-matched synthetic Sγ1 sequence to mediate substantial CSR to IgG1 in mutant B cells activated under conditions that stimulate IgG1 switching in WT B cells. We conclude that Sγ3 can function similarly to Sγ1 in mediating endogenous CSR to IgG1. The approach that we have developed will facilitate assays for IgH isotype–specific functions of other endogenous S regions.

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

10.1101/2020.11.25.398404 ◽

2020 ◽

Author(s):

Xuefei Zhang ◽

Hye Suk Yoon ◽

Aimee M. Chapdelaine-Williams ◽

Nia Kyritsis ◽

Frederick W. Alt

Keyword(s):

Chromosomal Rearrangements ◽

Regulatory Region ◽

Class Switch Recombination ◽

Physiological Role ◽

Ctcf Binding ◽

Dna Breaks ◽

Antibody Isotype ◽

Class Switch ◽

Igh Locus ◽

Antibody Class

ABSTRACTIgH class switch recombination (CSR) replaces Cμ constant region (CH) exons with one of six downstream CHS by joining transcription-targeted 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 ten 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 Sγ1. Moreover, deletion of all 3’IgH CBEs rendered the 6kb 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 a critical insulator for focusing loop extrusion-mediated 3’IgHRR transcriptional and CSR activities on upstream CH locus targets.SignificanceB lymphocytes change antibody heavy chain (IgH) isotypes by a recombination/deletion process called IgH class switch recombination (CSR). CSR involves introduction of DNA breaks into a donor switch (S) region and also into one of six downstream S regions, with joining of the breaks changing antibody isotype. A chromatin super-anchor, of unknown function, is located just downstream of the IgH locus. We show that complete deletion of this super-anchor variably decreases CSR to most S regions and creates an ectopic S region downstream of IgH locus that undergoes aberrant CSR-driven chromosomal rearrangements. Based on these and other findings, we conclude that the super-anchor downstream of IgH is a critical insulator for focusing potentially dangerous CSR rearrangements to the IgH locus.

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Multiple DSB Resection Activities Redundantly Promote Alternative End Joining-Mediated Class Switch Recombination

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.767624 ◽

2021 ◽

Vol 9 ◽

Author(s):

Xikui Sun ◽

Jingning Bai ◽

Jiejie Xu ◽

Xiaoli Xi ◽

Mingyu Gu ◽

...

Keyword(s):

High Throughput Sequencing ◽

Class Switch Recombination ◽

Class Switching ◽

End Joining ◽

Independent Manner ◽

Class Switch ◽

Alternative End Joining ◽

A Cell ◽

Non Homologous End Joining ◽

Antibody Class

Alternative end joining (A-EJ) catalyzes substantial level of antibody class switch recombination (CSR) in B cells deficient for classical non-homologous end joining, featuring increased switch (S) region DSB resection and junctional microhomology (MH). While resection has been suggested to initiate A-EJ in model DSB repair systems using engineered endonucleases, the contribution of resection factors to A-EJ-mediated CSR remains unclear. In this study, we systematically dissected the requirement for individual DSB resection factors in A-EJ-mediated class switching with a cell-based assay system and high-throughput sequencing. We show that while CtIP and Mre11 both are mildly required for CSR in WT cells, they play more critical roles in mediating A-EJ CSR, which depend on the exonuclease activity of Mre11. While DNA2 and the helicase/HRDC domain of BLM are required for A-EJ by mediating long S region DSB resection, in contrast, Exo1’s resection-related function does not play any obvious roles for class switching in either c-NHEJ or A-EJ cells, or mediated in an AID-independent manner by joining of Cas9 breaks. Furthermore, ATM and its kinase activity functions at least in part independent of CtIP/Mre11 to mediate A-EJ switching in Lig4-deficient cells. In stark contrast to Lig4 deficiency, 53BP1-deficient cells do not depend on ATM/Mre11/CtIP for residual joining. We discuss the roles for each resection factor in A-EJ-mediated CSR and suggest that the extent of requirements for resection is context dependent.

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SAMHD 1‐mediated dNTP degradation is required for efficient DNA repair during antibody class switch recombination

The EMBO Journal ◽

10.15252/embj.2019102931 ◽

2020 ◽

Vol 39 (15) ◽

Cited By ~ 1

Author(s):

Afzal Husain ◽

Jianliang Xu ◽

Hodaka Fujii ◽

Mikiyo Nakata ◽

Maki Kobayashi ◽

...

Keyword(s):

Dna Repair ◽

Class Switch Recombination ◽

Class Switch ◽

Antibody Class

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The Impact of Nuclear Organization and Homolgous Recombination in Repair of DNA Damage Introduced By Aid during Class Switch Recombination

Blood ◽

10.1182/blood.v124.21.2738.2738 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2738-2738

Author(s):

Pedro P Rocha ◽

Yi Fu ◽

JungHyun Kim ◽

Jane Skok

Keyword(s):

Cell Cycle ◽

Mouse Model ◽

B Cell ◽

Class Switch Recombination ◽

Enzyme Activation ◽

G1 Phase ◽

Repair Pathway ◽

Class Switch ◽

Igh Locus ◽

Pathway Choice

Abstract Class Switch Recombination (CSR) involves the introduction of double stranded breaks (DSBs) at the switch regions of the immunoglulin heavy chain (Igh) locus by the enzyme Activation Cytidine Deaminse (AID). AID can also act as a general mutator targeting other loci in the genome which can then either be repaired faithfully or in an error-prone fashion introducing mutations and potentially initiating B cell lymphoma. The factors contributing to the choice of repair pathway are not fully understood. Here we tested the hypothesis that repair pathway choice is influenced by differential accessibility and expression levels of target loci across cell cycle. More specifically in the context of CSR we tested whether differential regulation of gene accessibility across cell cycle is an important determinant for AID binding and subsequent repair pathway choice as different repair pathways predominate at different stages of cell cycle. Using 3D-FISH in conjunction with Immunofluorescence we observed that AID target genes that are faithfully repaired are more accessible (found in euchromatic regions) in the G2 phase of the cell cycle then genes that are frequently mutated. In contrast, those genes which are repaired in an error prone fashion are more accessible in the G1 phase of cell cycle. Since Homologous Recombination mediated repair (HR), which is a faithful repair mechanism, occurs in G2 we speculate that accessibility of these genes at this stage of cell cycle facilitates action by this repair pathway. Conversely, genes that are more accessible during the G1 phase of cell cycle will be repaired by the non-homologous end joining (NHEJ) repair pathway and therefore are more likely to be mutated. Thus, HR could be the pathway by which faithful repair is accomplished and use of the NHEJ pathway on the other hand could contribute to the introduction of dangerous DNA mutations that might lead to B cell transformation and cancer. To connect differences in accessibility with repair pathway usage, we used a mouse model carrying a hypomorphic mutation in BRCA2, a protein involved in HR. This is the first mouse model impaired in HR that eludes embryonic lethality and allows inspection of the role of this pathway in maintaining genomic stability in splenocytes undergoing CSR. Our preliminary investigations indicate that in Brca2 mutant B cells not only is the integrity of fathfully repaired loci compromised, but the Igh locus is also damaged. Taken together these results support our hypothesis and further indicate that the HR pathway is involved in repairing Igh. Given that approximately 95% of lymphomas are of B cell origin and many of these are associated with AID mediated breaks, it is crucial for us to understand the factors that influence targeting and repair. Disclosures No relevant conflicts of interest to declare.

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