scholarly journals Aberrant recombination and repair during immunoglobulin class switching in BRCA1-deficient human B cells

2015 ◽  
Vol 112 (7) ◽  
pp. 2157-2162 ◽  
Author(s):  
Andrea Björkman ◽  
Per Qvist ◽  
Likun Du ◽  
Margarita Bartish ◽  
Apostolos Zaravinos ◽  
...  
Keyword(s):  
Breast Cancer ◽  
B Cells ◽  
Class Switch Recombination ◽  
Cancer Type ◽  
End Joining ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Human B Cells ◽  
Breast Cancer Type

Breast cancer type 1 susceptibility protein (BRCA1) has a multitude of functions that contribute to genome integrity and tumor suppression. Its participation in the repair of DNA double-strand breaks (DSBs) during homologous recombination (HR) is well recognized, whereas its involvement in the second major DSB repair pathway, nonhomologous end-joining (NHEJ), remains controversial. Here we have studied the role of BRCA1 in the repair of DSBs in switch (S) regions during immunoglobulin class switch recombination, a physiological, deletion/recombination process that relies on the classical NHEJ machinery. A shift to the use of microhomology-based, alternative end-joining (A-EJ) and increased frequencies of intra-S region deletions as well as insertions of inverted S sequences were observed at the recombination junctions amplified from BRCA1-deficient human B cells. Furthermore, increased use of long microhomologies was found at recombination junctions derived from E3 ubiquitin-protein ligase RNF168-deficient, Fanconi anemia group J protein (FACJ, BRIP1)-deficient, or DNA endonuclease RBBP8 (CtIP)-compromised cells, whereas an increased frequency of S-region inversions was observed in breast cancer type 2 susceptibility protein (BRCA2)-deficient cells. Thus, BRCA1, together with its interaction partners, seems to play an important role in repairing DSBs generated during class switch recombination by promoting the classical NHEJ pathway. This may not only provide a general mechanism underlying BRCA1’s function in maintaining genome stability and tumor suppression but may also point to a previously unrecognized role of BRCA1 in B-cell lymphomagenesis.

scholarly journals Involvement of Artemis in nonhomologous end-joining during immunoglobulin class switch recombination

2008 ◽  
Vol 205 (13) ◽  
pp. 3031-3040 ◽  
Author(s):  
Likun Du ◽  
Mirjam van der Burg ◽  
Sergey W. Popov ◽  
Ashwin Kotnis ◽  
Jacques J.M. van Dongen ◽  
...  
Keyword(s):  
Class Switch Recombination ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Igg Subclass ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Human B Cells

DNA double-strand breaks (DSBs) introduced in the switch (S) regions are intermediates during immunoglobulin class switch recombination (CSR). These breaks are subsequently recognized, processed, and joined, leading to recombination of the two S regions. Nonhomologous end-joining (NHEJ) is believed to be the principle mechanism involved in DSB repair during CSR. One important component in NHEJ, Artemis, has however been considered to be dispensable for efficient CSR. In this study, we have characterized the S recombinational junctions from Artemis-deficient human B cells. Sμ–Sα junctions could be amplified from all patients tested and were characterized by a complete lack of “direct” end-joining and a remarkable shift in the use of an alternative, microhomology-based end-joining pathway. Sμ–Sγ junctions could only be amplified from one patient who carries “hypomorphic” mutations. Although these Sμ–Sγ junctions appear to be normal, a significant increase of an unusual type of sequential switching from immunoglobulin (Ig)M, through one IgG subclass, to a different IgG subclass was observed, and the Sγ–Sγ junctions showed long microhomologies. Thus, when the function of Artemis is impaired, varying modes of CSR junction resolution may be used for different S regions. Our findings strongly link Artemis to the predominant NHEJ pathway during CSR.

scholarly journals Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination

2018 ◽  
Vol 115 (34) ◽  
pp. 8615-8620 ◽  
Author(s):  
Jennifer L. Crowe ◽  
Zhengping Shao ◽  
Xiaobin S. Wang ◽  
Pei-Chi Wei ◽  
Wenxia Jiang ◽  
...  
Keyword(s):  
B Cells ◽  
High Throughput Sequencing ◽  
Class Switch Recombination ◽  
End Joining ◽  
Extensive Resection ◽  
Structural Role ◽  
Class Switch ◽  
Cell Class ◽  
Dependent Protein

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD, but not DNA-PKcs−/−, B cells. Meanwhile, the residual joints from DNA-PKcsKD/KD cells and the efficient Sµ-Sγ1 junctions from DNA-PKcs−/− B cells both display similar preferences for small (2–6 nt) microhomologies (MH). In DNA-PKcs−/− cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Sε and Sµ-Sµ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.

scholarly journals Immunoglobulin Class Switch Recombination Is Impaired in Atm-deficient Mice

2004 ◽  
Vol 200 (9) ◽  
pp. 1111-1121 ◽  
Author(s):  
Joanne M. Lumsden ◽  
Thomas McCarty ◽  
Lisa K. Petiniot ◽  
Rhuna Shen ◽  
Carrolee Barlow ◽  
...  
Keyword(s):  
B Cells ◽  
Dna Recombination ◽  
Class Switch Recombination ◽  
Intrinsic Defect ◽  
Wild Type ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Deficient Mice

Immunoglobulin class switch recombination (Ig CSR) involves DNA double strand breaks (DSBs) at recombining switch regions and repair of these breaks by nonhomologous end-joining. Because the protein kinase ataxia telengiectasia (AT) mutated (ATM) plays a critical role in DSB repair and AT patients show abnormalities of Ig isotype expression, we assessed the role of ATM in CSR by examining ATM-deficient mice. In response to T cell–dependent antigen (Ag), Atm−/− mice secreted substantially less Ag-specific IgA, IgG1, IgG2b, and IgG3, and less total IgE than Atm+/+ controls. To determine whether Atm−/− B cells have an intrinsic defect in their ability to undergo CSR, we analyzed in vitro responses of purified B cells. Atm−/− cells secreted substantially less IgA, IgG1, IgG2a, IgG3, and IgE than wild-type (WT) controls in response to stimulation with lipopolysaccharide, CD40 ligand, or anti-IgD plus appropriate cytokines. Molecular analysis of in vitro responses indicated that WT and Atm−/− B cells produced equivalent amounts of germline IgG1 and IgE transcripts, whereas Atm−/− B cells produced markedly reduced productive IgG1 and IgE transcripts. The reduction in isotype switching by Atm−/− B cells occurs at the level of genomic DNA recombination as measured by digestion–circularization PCR. Analysis of sequences at CSR sites indicated that there is greater microhomology at the μ–γ1 switch junctions in ATM B cells than in wild-type B cells, suggesting that ATM function affects the need or preference for sequence homology in the CSR process. These findings suggest a role of ATM in DNA DSB recognition and/or repair during CSR.

scholarly journals DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

2020 ◽  
Author(s):  
Jennifer L. Crowe ◽  
Xiaobin S. Wang ◽  
Zhengping Shao ◽  
Brian J. Lee ◽  
Verna Estes ◽  
...  
Keyword(s):  
Dna Repair ◽  
B Cells ◽  
Class Switch Recombination ◽  
Chromosomal Translocation ◽  
Radiation Sensitivity ◽  
Repair Pathway ◽  
End Joining ◽  
Class Switch ◽  
Pathway Choice

AbstractThe DNA-dependent protein kinase (DNA-PK), composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends, and recruits and activates DNA-PK. DNA-PKcs is the best-characterized substrate of DNA-PK, which phosphorylates DNA-PKcs at both the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity, suggesting a role of T2609 phosphorylation in DNA repair. Using the DNA-PKcs5A mouse model carrying an alanine substitution at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs5A/5A B cells reveal increased chromosomal translocation, excess end-resection, and preferential usage of micro-homology – all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.Key pointsLoss of T2069 cluster phosphorylation of DNA-PKcs promotes Alt-EJ-mediated CSR.

scholarly journals DNA-PKcs Is Involved in Ig Class Switch Recombination in Human B Cells

2015 ◽  
Vol 195 (12) ◽  
pp. 5608-5615 ◽  
Author(s):  
Andrea Björkman ◽  
Likun Du ◽  
Kerstin Felgentreff ◽  
Cornelia Rosner ◽  
Radhika Pankaj Kamdar ◽  
...  
Keyword(s):  
B Cells ◽  
Class Switch Recombination ◽  
Class Switch ◽  
Human B Cells

scholarly journals Pseudocell Tracer—A method for inferring dynamic trajectories using scRNAseq and its application to B cells undergoing immunoglobulin class switch recombination

2021 ◽  
Vol 17 (5) ◽  
pp. e1008094
Author(s):  
Derek Reiman ◽  
Godhev Kumar Manakkat Vijay ◽  
Heping Xu ◽  
Andrew Sonin ◽  
Dianyu Chen ◽  
...  
Keyword(s):  
B Cells ◽  
Class Switch Recombination ◽  
Cellular Heterogeneity ◽  
Supervised Machine Learning ◽  
Biological Information ◽  
Intermediate Cell ◽  
Dimensional Manifold ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Igg1 Isotype

Single cell RNA sequencing (scRNAseq) can be used to infer a temporal ordering of cellular states. Current methods for the inference of cellular trajectories rely on unbiased dimensionality reduction techniques. However, such biologically agnostic ordering can prove difficult for modeling complex developmental or differentiation processes. The cellular heterogeneity of dynamic biological compartments can result in sparse sampling of key intermediate cell states. To overcome these limitations, we develop a supervised machine learning framework, called Pseudocell Tracer, which infers trajectories in pseudospace rather than in pseudotime. The method uses a supervised encoder, trained with adjacent biological information, to project scRNAseq data into a low-dimensional manifold that maps the transcriptional states a cell can occupy. Then a generative adversarial network (GAN) is used to simulate pesudocells at regular intervals along a virtual cell-state axis. We demonstrate the utility of Pseudocell Tracer by modeling B cells undergoing immunoglobulin class switch recombination (CSR) during a prototypic antigen-induced antibody response. Our results revealed an ordering of key transcription factors regulating CSR to the IgG1 isotype, including the concomitant expression of Nfkb1 and Stat6 prior to the upregulation of Bach2 expression. Furthermore, the expression dynamics of genes encoding cytokine receptors suggest a poised IL-4 signaling state that preceeds CSR to the IgG1 isotype.

scholarly journals IL-21 is a broad negative regulator of IgE class switch recombination in mouse and human B cells

2020 ◽  
Vol 217 (5) ◽  
Author(s):  
Zhiyong Yang ◽  
Chung-An M. Wu ◽  
Sasha Targ ◽  
Christopher D.C. Allen
Keyword(s):  
B Cells ◽  
Class Switch Recombination ◽  
Primary Source ◽  
Negative Regulator ◽  
Class Switch ◽  
Cellular Analysis ◽  
Follicular Helper ◽  
Human B Cells

IgE antibodies may elicit potent allergic reactions, and their production is tightly controlled. The tendency to generate IgE has been thought to reflect the balance between type 1 and type 2 cytokines, with the latter promoting IgE. Here, we reevaluated this paradigm by a direct cellular analysis, demonstrating that IgE production was not limited to type 2 immune responses yet was generally constrained in vivo. IL-21 was a critical negative regulator of IgE responses, whereas IFN-γ, IL-6, and IL-10 were dispensable. Follicular helper T cells were the primary source of IL-21 that inhibited IgE responses by directly engaging the IL-21 receptor on B cells and triggering STAT3-dependent signaling. We reconciled previous discordant results between mouse and human B cells and revealed that the inhibition of IgE class switch recombination by IL-21 was attenuated by CD40 signaling, whereas IgG1 class switch recombination was potentiated by IL-21 in the context of limited IL-4. These findings establish key features of the extrinsic regulation of IgE production by cytokines.

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