scholarly journals Histone methyltransferase MMSET promotes AID-mediated DNA breaks at the donor switch region during class switch recombination

2017 ◽  
Vol 114 (49) ◽  
pp. E10560-E10567 ◽  
Author(s):  
Hai Vu Nguyen ◽  
Junchao Dong ◽  
Rohit A. Panchakshari ◽  
Vipul Kumar ◽  
Frederick W. Alt ◽  
...  
Keyword(s):  
B Cells ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Histone Methyltransferase ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Set Domain ◽  
End Joining ◽  
Class Switch

In B cells, Ig class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), the activity of which leads to DNA double-strand breaks (DSBs) within IgH switch (S) regions. Preferential targeting of AID-mediated DSBs to S sequences is critical for allowing diversification of antibody functions, while minimizing potential off-target oncogenic events. Here, we used gene targeted inactivation of histone methyltransferase (HMT) multiple myeloma SET domain (MMSET) in mouse B cells and the CH12F3 cell line to explore its role in CSR. We find that deletion of MMSET-II, the isoform containing the catalytic SET domain, inhibits CSR without affecting either IgH germline transcription or joining of DSBs within S regions by classical nonhomologous end joining (C-NHEJ). Instead, we find that MMSET-II inactivation leads to decreased AID recruitment and DSBs at the upstream donor Sμ region. Our findings suggest a role for the HMT MMSET in promoting AID-mediated DNA breaks during CSR.

scholarly journals The cohesin complex regulates immunoglobulin class switch recombination

2013 ◽  
Vol 210 (12) ◽  
pp. 2495-2502 ◽  
Author(s):  
Anne-Sophie Thomas-Claudepierre ◽  
Ebe Schiavo ◽  
Vincent Heyer ◽  
Marjorie Fournier ◽  
Adeline Page ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Nonhomologous End Joining ◽  
Cohesin Complex ◽  
Dna Breaks ◽  
End Joining ◽  
Regulatory Subunits ◽  
Class Switch ◽  
Chromatid Cohesion ◽  
Switch Regions

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to switch regions and by the subsequent generation of double-stranded DNA breaks (DSBs). These DNA breaks are ultimately resolved through the nonhomologous end joining (NHEJ) pathway. We show that during CSR, AID associates with subunits of cohesin, a complex previously implicated in sister chromatid cohesion, DNA repair, and the formation of DNA loops between enhancers and promoters. Furthermore, we implicate the cohesin complex in the mechanism of CSR by showing that cohesin is dynamically recruited to the Sμ-Cμ region of the IgH locus during CSR and that knockdown of cohesin or its regulatory subunits results in impaired CSR and increased usage of microhomology-based end joining.

scholarly journals BRCT-domain protein BRIT1 influences class switch recombination

2017 ◽  
Vol 114 (31) ◽  
pp. 8354-8359 ◽  
Author(s):  
Wei-Feng Yen ◽  
Ashutosh Chaudhry ◽  
Bharat Vaidyanathan ◽  
William T. Yewdell ◽  
Joseph N. Pucella ◽  
...  
Keyword(s):  
B Cells ◽  
Ex Vivo ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
B Cell Lymphoma ◽  
Brct Domain ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Class Switch ◽  
Domain Protein

DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs.

scholarly journals DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination

2008 ◽  
Vol 205 (3) ◽  
pp. 557-564 ◽  
Author(s):  
Sonia Franco ◽  
Michael M. Murphy ◽  
Gang Li ◽  
Tiffany Borjeson ◽  
Cristian Boboila ◽  
...  
Keyword(s):  
B Cells ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch ◽  
Chromosomal Breaks ◽  
Dependent Protein ◽  
Activation Conditions

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Artemis are classical nonhomologous DNA end-joining (C-NHEJ) factors required for joining a subset of DNA double-strand breaks (DSB), particularly those requiring end processing. In mature B cells, activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) by introducing lesions into S regions upstream of two recombining CH exons, which are processed into DSBs and rejoined by C-NHEJ to complete CSR. The function of DNA-PKcs in CSR has been controversial with some reports but not others showing that DNA-PKcs–deficient mice are significantly impaired for CSR. Artemis-deficient B cells reportedly undergo CSR at normal levels. Overall, it is still not known whether there are any CSR-associated DSBs that require DNA-PKcs and/or Artemis to be joined. Here, we have used an immunoglobulin (Ig)H locus-specific fluorescent in situ hybridization assay to unequivocally demonstrate that both DNA-PKcs and, unexpectedly, Artemis are necessary for joining a subset of AID-dependent DSBs. In the absence of either factor, B cells activated for CSR frequently generate AID-dependent IgH locus chromosomal breaks and translocations. We also find that under specific activation conditions, DNA-PKcs−/− B cells with chromosomal breaks are eliminated or at least prevented from progressing to metaphase via a p53-dependent response.

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 An in vivo study of the impact of deficiency in the DNA repair proteins PAXX and XLF on development and maturation of the hemolymphoid system

2020 ◽  
Vol 295 (8) ◽  
pp. 2398-2406 ◽  
Author(s):  
Stefania Musilli ◽  
Vincent Abramowski ◽  
Benoit Roch ◽  
Jean-Pierre de Villartay
Keyword(s):  
Class Switch Recombination ◽  
Adaptive Immune System ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
Hematopoietic Progenitors ◽  
End Joining ◽  
Class Switch ◽  
A Genome ◽  
The Impact

Repair of DNA double-strand breaks by the nonhomologous end joining pathway is central for proper development of the adaptive immune system. This repair pathway involves eight factors, including XRCC4-like factor (XLF)/Cernunnos and the paralog of XRCC4 and XLF, PAXX nonhomologous end joining factor (PAXX). Xlf−/− and Paxx−/− mice are viable and exhibit only a mild immunophenotype. However, mice lacking both PAXX and XLF are embryonic lethal because postmitotic neurons undergo massive apoptosis in embryos. To decipher the roles of PAXX and XLF in both variable, diversity, and joining recombination and immunoglobulin class switch recombination, here, using Cre/lox-specific deletion to prevent double-KO embryonic lethality, we developed two mouse models of a conditional Xlf KO in a Paxx−/− background. Cre expressed under control of the iVav or CD21 promoter enabled Xlf deletion in early hematopoietic progenitors and splenic mature B cells, respectively. We demonstrate the XLF and PAXX interplay during variable, diversity, and joining recombination in vivo but not during class switch recombination, for which PAXX appeared to be fully dispensable. Xlf/Paxx double KO in hematopoietic progenitors resulted in a shorter lifespan associated with onset of thymic lymphomas, revealing a genome caretaking function of XLF/PAXX.

scholarly journals Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome

2020 ◽  
Vol 217 (11) ◽  
Author(s):  
Angela Helfricht ◽  
Peter E. Thijssen ◽  
Magdalena B. Rother ◽  
Rashmi G. Shah ◽  
Likun Du ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Class Switch Recombination ◽  
Nonhomologous End Joining ◽  
Dna Breaks ◽  
End Joining ◽  
Immunoglobulin Production ◽  
Icf Syndrome ◽  
Class Switch ◽  
Centromeric Instability ◽  
Gene Defects

The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates its auto-poly(ADP-ribosyl)ation. The zinc-finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, through its association with poly(ADP-ribose) chains, ZBTB24 protects them from degradation by poly(ADP-ribose) glycohydrolase (PARG). This facilitates the poly(ADP-ribose)-dependent assembly of the LIG4/XRCC4 complex at DNA breaks, thereby promoting error-free NHEJ. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF2 syndrome.

scholarly journals Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4

2010 ◽  
Vol 207 (2) ◽  
pp. 417-427 ◽  
Author(s):  
Cristian Boboila ◽  
Catherine Yan ◽  
Duane R. Wesemann ◽  
Mila Jankovic ◽  
Jing H. Wang ◽  
...  
Keyword(s):  
B Cells ◽  
Class Switch Recombination ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Dsb Repair ◽  
Class Switch ◽  
Dna Double Strand Break ◽  
Alternative End Joining

The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; “MH-mediated” joins) or no homologies (“direct” joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via “alternative” end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR.

scholarly journals Parp1 facilitates alternative NHEJ, whereas Parp2 suppresses IgH/c-myc translocations during immunoglobulin class switch recombination

2009 ◽  
Vol 206 (5) ◽  
pp. 1047-1056 ◽  
Author(s):  
Isabelle Robert ◽  
Françoise Dantzer ◽  
Bernardo Reina-San-Martin
Keyword(s):  
Dna Damage ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dependent Manner ◽  
Dna Breaks ◽  
End Joining ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Switch Regions ◽  
Alternative Nhej

Immunoglobulin class switch recombination (CSR) is initiated by DNA breaks triggered by activation-induced cytidine deaminase (AID). These breaks activate DNA damage response proteins to promote appropriate repair and long-range recombination. Aberrant processing of these breaks, however, results in decreased CSR and/or increased frequency of illegitimate recombination between the immunoglobulin heavy chain locus and oncogenes like c-myc. Here, we have examined the contribution of the DNA damage sensors Parp1 and Parp2 in the resolution of AID-induced DNA breaks during CSR. We find that although Parp enzymatic activity is induced in an AID-dependent manner during CSR, neither Parp1 nor Parp2 are required for CSR. We find however, that Parp1 favors repair of switch regions through a microhomology-mediated pathway and that Parp2 actively suppresses IgH/c-myc translocations. Thus, we define Parp1 as facilitating alternative end-joining and Parp2 as a novel translocation suppressor during CSR.

scholarly journals 53BP1 regulates DNA resection and the choice between classical and alternative end joining during class switch recombination

2010 ◽  
Vol 207 (4) ◽  
pp. 855-865 ◽  
Author(s):  
Anne Bothmer ◽  
Davide F. Robbiani ◽  
Niklas Feldhahn ◽  
Anna Gazumyan ◽  
Andre Nussenzweig ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Response Factors ◽  
Class Switch ◽  
Damage Response ◽  
Switch Regions

Class switch recombination (CSR) diversifies antibodies by joining highly repetitive DNA elements, which are separated by 60–200 kbp. CSR is initiated by activation-induced cytidine deaminase, an enzyme that produces multiple DNA double-strand breaks (DSBs) in switch regions. Switch regions are joined by a mechanism that requires an intact DNA damage response and classical or alternative nonhomologous end joining (A-NHEJ). Among the DNA damage response factors, 53BP1 has the most profound effect on CSR. We explore the role of 53BP1 in intrachromosomal DNA repair using I-SceI to introduce paired DSBs in the IgH locus. We find that the absence of 53BP1 results in an ataxia telangiectasia mutated–dependent increase in DNA end resection and that resected DNA is preferentially repaired by microhomology-mediated A-NHEJ. We propose that 53BP1 favors long-range CSR in part by protecting DNA ends against resection, which prevents A-NHEJ–dependent short-range rejoining of intra–switch region DSBs.

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