Switch tandem repeats influence the choice of the alternative end-joining pathway in class switch recombination
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE174296 | GEO | 2022/05/11
REPOSITORIES: GEO
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Project description:Switch tandem repeats influence the choice of the alternative end-joining pathway in class switch recombination
| PRJNA729240 | ENA
Project description:Immunoglobulin class switch recombination (CSR) plays an important role in humoral imm\une responses by changing the effector functions of antibodies. CSR occurs between highly repetitive switch (S) sequences located upstream of immunoglobulin constant gene exons. Switch sequences differ in size, the nature of their repeats, and the density of the motifs targeted by the activation-induced cytidine deaminase (AID), the enzyme that initiates CSR. CSR involves double-strand breaks (DSBs) at the universal Sµ donor region and one of the acceptor S regions. The DSBs ends are fused by the classical non-homologous end-joining (C-NHEJ) and the alternative-NHEJ (A-NHEJ) pathways. Of the two pathways, the A-NHEJ displays a bias towards longer junctional micro-homologies (MHs). The Sµ region displays features that distinguish it from other S regions, but the molecular basis of Sµ specificity is ill-understood. We used a mouse line in which the downstream Sγ3 region was put under the control of the Eµ enhancer, which regulates Sµ, and analyzed its recombination activity by CSR-HTGTS. Here, we show that provision of Eµ enhancer to Sγ3 is sufficient to confer the recombinational features of Sµ to Sγ3, including efficient AID recruitment, enhanced internal deletions and robust donor function in CSR. Moreover, junctions involving Sγ3 display a bias for longer MH irrespective of sequence homology with switch acceptor sites. The data suggest that the propensity for increased MH usage is an intrinsic property of Sγ3 sequence, and that the tandem repeats of the donor site influence the choice of the A-NHEJ.
| S-EPMC9149575 | biostudies-literature
Project description:To generate antibodies with different effector functions, B cells undergo Immunoglobulin class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical non-homologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ~25% CSR can be achieved by the alternative end-joining (A-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ mediated repair of endonuclease generated breaks requires DNA end-resection, we show that CtIP-mediated DNA end-resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppress resection without altering the MH-pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ mediated CSR by suppressing inter-chromosomal translocations independent of end-resection. Finally, temperol analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favoriate substrates for MH-mediated end-joining contributing to the robustness and resection-indepndence of A-EJ-mediated CSR.
2020-09-18 | GSE156392 | GEO
Project description:IgH class switch recombination (CSR) in B lymphocytes switches IgH constant regions to change antibody functions. CSR is initiated by DNA double strand breaks (DSBs) within a donor IgH switch (S) region and a downstream acceptor S region. CSR is completed by fusing donor and acceptor S region DSB ends by classical non-homologous end-joining (C-NHEJ) and, in its absence, by alternative end-joining (A-EJ) that is more biased to use longer junctional micro-homologies (MHs). Deficiency for DSB response (DSBR) factors, including ATM and 53BP1, variably impair CSR end-joining, with 53BP1 deficiency having the greatest impact. However, studies of potential impact of DSBR factor deficiencies on MH-mediated CSR end-joining have been technically limited. We now use a robust DSB joining assay to elucidate impacts of deficiencies for DSBR factors on CSR and chromosomal translocation junctions in primary mouse B cells and CH12F3 B lymphoma cells. Compared to wild-type, CSR and c-Myc to S region translocation junctions in the absence of 53BP1, and to a lesser extent other DSBR factors, have increased MH-utilization; indeed, 53BP1-deficient MH-profiles resemble those associated with C-NHEJ deficiency. Yet, translocation junctions between c-Myc DSB and general DSBs genome-wide are not MH-biased in ATM-deficient versus wild-type CH12F3 cells and less biased in 53BP1- and C-NHEJ-deficient cells than CSR junctions or c-Myc to S region translocation junctions. We discuss potential roles of DSBR factors in suppressing increased MH-mediated DSB end-joining and features of S regions that may render their DSBs prone to MH-biased end-joining in the absence of DSBR factors.
2018-01-08 | GSE106922 | GEO
Project description:The autosomal recessive immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite recent successes in the identification of the underlying gene defects, it is currently 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 ICF2 patients impairs non-homologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and subtype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates auto-poly(ADP-ribosyl)ation of this enzyme. The zinc finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, by binding to poly(ADP-ribose) chains ZBTB24 protects these moieties from degradation by poly(ADP-ribose) glycohydrolase (PARG). This enhances the poly(ADP-ribose)-dependent interaction between PARP1 and the LIG4/XRCC4 NHEJ complex and promotes NHEJ by facilitating the assembly of this repair complex at DNA breaks. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF syndrome.
2020-08-19 | PXD014741 | Pride
Project description:CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining
| PRJNA657760 | ENA
Project description:The DNA-dependent protein kinase (DNA-PK), which is 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. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs5A mouse model carrying alanine substitutions 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 translocations, extensive use of distal switch regions (consistent with 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.
2020-07-18 | GSE154210 | GEO
Project description:Immunoglobulin heavy chain (Igh locus) class-switch recombination (CSR) requires targeted introduction of DNA double strand breaks (DSBs) into repetitive 'switch'-region DNA elements in the Igh locus and subsequent ligation between distal DSBs. Both canonical nonhomologous end joining (C-NHEJ) that seals DNA ends with little or no homology and a poorly defined alternative end joining (A-NHEJ, also known as alt-NHEJ) process that requires microhomology ends for ligation have been implicated in CSR. Here, we show that the DNA end-processing factor CtIP is required for microhomology-directed A-NHEJ during CSR. Additionally, we demonstrate that microhomology joins that are enriched upon depletion of the C-NHEJ component Ku70 require CtIP. Finally, we show that CtIP binds to switch-region DNA in a fashion dependent on activation-induced cytidine deaminase. Our results establish CtIP as a bona fide component of microhomology-dependent A-NHEJ and unmask a hitherto unrecognized physiological role of microhomology-mediated end joining in a C-NHEJ-proficient environment.
| S-EPMC3471154 | biostudies-literature
Project description: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. Smu-Salpha 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. Smu-Sgamma junctions could only be amplified from one patient who carries "hypomorphic" mutations. Although these Smu-Sgamma 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 Sgamma-Sgamma 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.
| S-EPMC2605234 | biostudies-literature
Project description:53BP1 is a DNA damage protein that forms phosphorylated H2AX (?-H2AX) dependent foci in a 1 Mb region surrounding DNA double-strand breaks (DSBs). In addition, 53BP1 promotes genomic stability by regulating the metabolism of DNA ends. We have compared the joining rates of paired DSBs separated by 1.2 kb to 27 Mb on chromosome 12 in the presence or absence of 53BP1. 53BP1 facilitates joining of intrachromosomal DSBs but only at distances corresponding to ?-H2AX spreading. In contrast, DNA end protection by 53BP1 is distance independent. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation are all required for DNA end protection and joining as measured by immunoglobulin class switch recombination. These data elucidate the molecular events that are required for 53BP1 to maintain genomic stability and point to a model wherein 53BP1 and H2AX cooperate to repress resection of DSBs.
| S-EPMC3142663 | biostudies-literature