scholarly journals ATM Is Required for Efficient Recombination between Immunoglobulin Switch Regions

2004 ◽  
Vol 200 (9) ◽  
pp. 1103-1110 ◽  
Author(s):  
Bernardo Reina-San-Martin ◽  
Hua Tang Chen ◽  
André Nussenzweig ◽  
Michel C. Nussenzweig
Keyword(s):  
Somatic Hypermutation ◽  
Serum Antibody ◽  
Strand Break ◽  
Cell Cycle Checkpoints ◽  
Immunoglobulin Gene ◽  
Switch Region ◽  
Atm Kinase ◽  
Class Switch ◽  
Gene Diversification ◽  
Switch Regions

Ataxia telangiectasia mutated (ATM) kinase is critical for initiating the signaling pathways that lead to cell cycle checkpoints and DNA double strand break repair. In the absence of ATM, humans and mice show a primary immunodeficiency that includes low serum antibody titers, but the role of ATM in antigen-driven immunoglobulin gene diversification has not been defined. Here, we show that although ATM is dispensable for somatic hypermutation, it is required for efficient class switch recombination (CSR). The defect in CSR is not due to alterations in switch region transcription, accessibility, DNA damage checkpoint protein recruitment, or short-range intra-switch region recombination. Only long-range inter-switch recombination is defective, indicating an unexpected role for ATM in switch region synapsis during CSR.

scholarly journals Immunoglobulin Class Switch Recombination Is Initiated by Rare Cytosine Deamination Events at Switch Regions

2020 ◽  
Vol 40 (16) ◽  
Author(s):  
Ahrom Kim ◽  
Li Han ◽  
Kefei Yu
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Strand Break ◽  
Switch Region ◽  
Cytosine Deamination ◽  
Class Switch ◽  
Switch Regions ◽  
Dna Strands ◽  
Uracil Repair

ABSTRACT Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) class switch recombination (CSR), somatic hypermutation (SHM), and gene conversion by converting DNA cytosines to uracils at specific genomic regions. In this study, we examined AID footprints across the entire length of an engineered switch region in cells ablated for uracil repair. We found that AID deamination occurs predominantly at WRC hot spots (where W is A or T and R is A or G) and that the deamination frequency remains constant across the entire switch region. Importantly, we analyzed monoallelic AID deamination footprints on both DNA strands occurring within a single cell cycle. We found that AID generates few and mostly isolated uracils in the switch region, although processive AID deaminations are evident in some molecules. The frequency of molecules containing deamination on both DNA strands at the acceptor switch region correlates with the class switch efficiency, raising the possibility that the minimal requirement for DNA double-strand break (DSB) formation is as low as even one AID deamination event on both DNA strands.

scholarly journals H2AX Is Required for Recombination Between Immunoglobulin Switch Regions but Not for Intra-Switch Region Recombination or Somatic Hypermutation

2003 ◽  
Vol 197 (12) ◽  
pp. 1767-1778 ◽  
Author(s):  
Bernardo Reina-San-Martin ◽  
Simone Difilippantonio ◽  
Leif Hanitsch ◽  
Revati F. Masilamani ◽  
André Nussenzweig ◽  
...  
Keyword(s):  
B Cells ◽  
Posttranslational Modifications ◽  
Somatic Hypermutation ◽  
Dna Lesions ◽  
Switch Region ◽  
Histone H2ax ◽  
Class Switch ◽  
Switch Regions ◽  
Precise Role

Changes in chromatin structure induced by posttranslational modifications of histones are important regulators of genomic function. Phosphorylation of histone H2AX promotes DNA repair and helps maintain genomic stability. Although B cells lacking H2AX show impaired class switch recombination (CSR), the precise role of H2AX in CSR and somatic hypermutation (SHM) has not been defined. We show that H2AX is not required for SHM, suggesting that the processing of DNA lesions leading to SHM is fundamentally different from CSR. Impaired CSR in H2AX−/− B cells is not due to alterations in switch region transcription, accessibility, or aberrant joining. In the absence of H2AX, short-range intra-switch region recombination proceeds normally while long-range inter-switch region recombination is impaired. Our results suggest a role for H2AX in regulating the higher order chromatin remodeling that facilitates switch region synapsis.

scholarly journals Fine-Structure Analysis of Activation-Induced Deaminase Accessibility to Class Switch Region R-Loops

2005 ◽  
Vol 25 (5) ◽  
pp. 1730-1736 ◽  
Author(s):  
Kefei Yu ◽  
Deepankar Roy ◽  
Melina Bayramyan ◽  
Ian S. Haworth ◽  
Michael R. Lieber
Keyword(s):  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Site Preference ◽  
Switch Region ◽  
Class Switch ◽  
High Resolution Analysis ◽  
Switch Regions ◽  
Resolution Analysis ◽  
Activation Induced Deaminase ◽  
Dna Strand

ABSTRACT Activation-induced deaminase (AID) is essential for class switch recombination and somatic hypermutation, and it has the ability to deaminate single-stranded DNA at cytidines. Mammalian class switch regions form R-loops upon transcription in the physiological orientation. The displaced DNA strand of an R-loop is forced to wrap around the RNA-DNA hybrid; hence, it may not have complete exposure to proteins. A fundamental question concerns the extent to which AID is accessible to the displaced strand of a transcription-generated R-loop. We used a minimal R-loop to carry out high-resolution analysis of the precise locations of AID action. We found that AID deaminates on the displaced DNA strand across the entire length of the R-loop. Displaced strand locations with a WRC (where W is A or T and R is A or G) sequence are preferred targets, but there are clear exceptions. These WRC deviations may be due to steric constraints on the accessibility of AID to these sites as the displaced strand twists around the RNA-DNA duplex. This phenomenon may explain the lack of WRC site preference at the mutations surrounding class switch recombination junctions.

scholarly journals Intrinsic Strand-Incision Activity of Human UNG: Implications for Nick Generation in Immunoglobulin Gene Diversification

2021 ◽  
Vol 12 ◽  
Author(s):  
Marina Alexeeva ◽  
Marivi Nabong Moen ◽  
Xiang Ming Xu ◽  
Anette Rasmussen ◽  
Ingar Leiros ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Excision Repair ◽  
Cytidine Deaminase ◽  
Strand Break ◽  
Immunoglobulin Gene ◽  
Dna Double Strand Breaks ◽  
Cytosine Deamination ◽  
Considerable Uncertainty ◽  
Gene Diversification ◽  
Cellular Dna

Uracil arises in cellular DNA by cytosine (C) deamination and erroneous replicative incorporation of deoxyuridine monophosphate opposite adenine. The former generates C → thymine transition mutations if uracil is not removed by uracil-DNA glycosylase (UDG) and replaced by C by the base excision repair (BER) pathway. The primary human UDG is hUNG. During immunoglobulin gene diversification in activated B cells, targeted cytosine deamination by activation-induced cytidine deaminase followed by uracil excision by hUNG is important for class switch recombination (CSR) and somatic hypermutation by providing the substrate for DNA double-strand breaks and mutagenesis, respectively. However, considerable uncertainty remains regarding the mechanisms leading to DNA incision following uracil excision: based on the general BER scheme, apurinic/apyrimidinic (AP) endonuclease (APE1 and/or APE2) is believed to generate the strand break by incising the AP site generated by hUNG. We report here that hUNG may incise the DNA backbone subsequent to uracil excision resulting in a 3´-α,β-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5´-phosphate. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2´ occurs via the formation of a C1´ enolate intermediate. UIP is removed from the 3´-end by hAPE1. This shows that the first two steps in uracil BER can be performed by hUNG, which might explain the significant residual CSR activity in cells deficient in APE1 and APE2.

scholarly journals AID from bony fish catalyzes class switch recombination

2005 ◽  
Vol 202 (6) ◽  
pp. 733-738 ◽  
Author(s):  
Vasco M. Barreto ◽  
Qiang Pan-Hammarstrom ◽  
Yaofeng Zhao ◽  
Lennart Hammarstrom ◽  
Ziva Misulovin ◽  
...  
Keyword(s):  
B Cells ◽  
Catalytic Domain ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Adaptive Immune System ◽  
Bony Fish ◽  
Class Switching ◽  
Class Switch ◽  
Switch Regions

Class switch recombination was the last of the lymphocyte-specific DNA modification reactions to appear in the evolution of the adaptive immune system. It is absent in cartilaginous and bony fish, and it is common to all tetrapods. Class switching is initiated by activation-induced cytidine deaminase (AID), an enzyme expressed in cartilaginous and bony fish that is also required for somatic hypermutation. Fish AID differs from orthologs found in tetrapods in several respects, including its catalytic domain and carboxy-terminal region, both of which are essential for the switching reaction. To determine whether evolution of class switch recombination required alterations in AID, we assayed AID from Japanese puffer and zebra fish for class-switching activity in mouse B cells. We find that fish AID catalyzes class switch recombination in mammalian B cells. Thus, AID had the potential to catalyze this reaction before the teleost and tetrapod lineages diverged, suggesting that the later appearance of a class-switching reaction was dependent on the evolution of switch regions and multiple constant regions in the IgH locus.

scholarly journals Frequency of immunoglobulin E class switching is autonomously determined and independent of prior switching to other classes.

1994 ◽  
Vol 179 (6) ◽  
pp. 2023-2026 ◽  
Author(s):  
S Jung ◽  
G Siebenkotten ◽  
A Radbruch
Keyword(s):  
Immunoglobulin E ◽  
Low Frequency ◽  
Switch Region ◽  
Major Fraction ◽  
Class Switching ◽  
Class Switch ◽  
Inherent Feature ◽  
Functional Implications ◽  
Switch Regions ◽  
Sequential Switching

Both, in humans and in mice, a major fraction of immunoglobulin E (IgE)-expressing B lymphocytes develops by sequential Ig class switching from IgM via IgG to IgE. This sequential class switch might have functional implications for the frequency and repertoire of IgE+ cells. Here we show that in mutant mice, in which sequential switching to IgE via IgG1 is blocked, the frequency of cells switching to IgE is not affected. Thus, sequential class switching to IgE merely reflects the simultaneous accessibility of two acceptor switch regions for switch recombination, induced by one cytokine, but with markedly distinct efficiency. Analysis of switch recombination on both IgH alleles of switched cells shows that the low frequency of switching to IgE is an inherent feature of the S epsilon switch region and its control elements.

Regulation of ϵ germline transcription and switch region mutations by IgH locus 3′ enhancers in transgenic mice

Blood ◽  
2006 ◽  
Vol 109 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Jurga Laurencikiene ◽  
Vytas Tamosiunas ◽  
Eva Severinson
Keyword(s):  
B Cells ◽  
Cytidine Deaminase ◽  
Switch Region ◽  
Endogenous Gene ◽  
Class Switch ◽  
Germline Transcription ◽  
Igh Locus ◽  
Activation Induced Cytidine Deaminase ◽  
Switch Regions ◽  
Transgenic Lines

Abstract Germline (GL) transcription is regulated by specific promoters and immunoglobulin heavy chain (IgH) 3′ locus enhancers and is necessary for Ig class-switch recombination (CSR). We have generated different transgenic lines containing the GL ϵ promoter, switch (S) ϵ region, and constant (C) ϵ region with or without the DNase I–sensitive regions (HS) 3A-HS1,2 or HS3B-HS4 3′ IgH enhancer pairs. The enhancerless construct was expressed in B cells activated by interleukin (IL)–4 and CD40, thus resembling regulation of the endogenous gene. Both enhancer-containing transgenes efficiently increased expression in B cells and were strongly up-regulated by stimuli. In addition, Sϵ regions of the transgene containing HS3B-HS4 were mutated in activated, sorted B cells. Such mutations are known to precede CSR and are dependent on activation-induced cytidine deaminase (AID). Our findings show that all elements necessary for recruitment of the recombination machinery are present in the transgene containing HS3 and HS4. These enhancers probably provide something more specific than mere increased accessibility of switch regions. We propose that transcription factors binding the enhancers help to target the recombination machinery to the switch regions.

scholarly journals IgH 3’ regulatory region increases ectopic class switch recombination

PLoS Genetics ◽  
2021 ◽  
Vol 17 (2) ◽  
pp. e1009288
Author(s):  
Sandrine Le Noir ◽  
Amélie Bonaud ◽  
Bastien Hervé ◽  
Audrey Baylet ◽  
François Boyer ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Regulatory Region ◽  
Class Switch Recombination ◽  
Dna Lesions ◽  
Reporter System ◽  
Long Distance ◽  
Class Switch ◽  
Super Enhancer ◽  
Switch Regions ◽  
Activation Induced Deaminase

DNA lesions inflicted by activation-induced deaminase (AID) instrumentally initiate the processes reshaping immunoglobulin genes in mature B-cells, from local somatic hypermutation (SHM) to junctions of distant breaks during class switch recombination (CSR). It remains incompletely understood how these divergent outcomes of AID attacks are differentially and temporally focused, with CSR strictly occurring in the Ig heavy chain (IgH) locus while SHM concentrates on rearranged V(D)J regions in the IgH and Ig light chain loci. In the IgH locus, disruption of either the 3’Regulatory Region (3’RR) super-enhancer or of switch (S) regions preceding constant genes, profoundly affects CSR. Reciprocally, we now examined if these elements are sufficient to induce CSR in a synthetic locus based on the Igκ locus backbone. Addition of a surrogate “core 3’RR” (c3’RR) and of a pair of transcribed and spliced Switch regions, together with a reporter system for “κ-CSR” yielded a switchable Igκ locus. While the c3’RR stimulated SHM at S regions, it also lowered the local SHM threshold necessary for switch recombination to occur. The 3’RR thus both helps recruit AID to initiate DNA lesions, but then also promotes their resolution through long-distance synapses and recombination following double-strand breaks.

scholarly journals Mlh1 Can Function in Antibody Class Switch Recombination Independently of Msh2

2003 ◽  
Vol 197 (10) ◽  
pp. 1377-1383 ◽  
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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