In vitro Naïve B-cell Class Switching Stimulation with ATM-inhibitor and DNA-PKcs-inhibitor
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PROVIDER: PRJNA935746 | ENA |
REPOSITORIES: ENA
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Project description:Analyze Class switch recombination of naïve B-cells in vitro mimicking Ataxia Telangiectasia using a small molecule inhibitor (KU-55933). We measure switched IgG and IgG memory B-cells, and analyzed transcriptomics (this data).
2024-05-21 | GSE225461 | GEO
Project description: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/KDcells 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-Se 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.
2018-07-26 | GSE117628 | GEO
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:In depth analysis of double strand break signaling was performed using mouse Pre-B cells and Human HCT116 cells. Our analysis included the induction of double strand breaks with ionizing radiation with the addition of ATM and DNA-PKcs inhibitors, alone and in combination.
2024-10-17 | PXD042258 | Pride
Project description:Although immunoglobulin class-switching from IgM to IgG enhances B cell receptor (BCR) signalling and antibody responses, certain subsets of B cell lymphomas retain unswitched IgM BCRs despite signs of class-switching in the immunoglobulin heavy chain loci. This suggests that expression of the IgG BCR suppresses progression of specific lymphoma subsets. We found that inducing BCR class switching from IgM to IgG1 or IgG4 inhibits proliferation of B EZB DLBCL lymphoma cell lines. To understand changes associated with class-switching we compared transcritional profiles of isogenic WSU-FSCCL cell lines artificially class-switched to express IgM and IgG BCRs.
2024-05-15 | GSE267440 | GEO
Project description:The classical non-homologous end-joining (cNHEJ) pathway is a major DNA double-strand break repair pathway in mammalian cells and is required for lymphocyte development and maturation. The DNA-dependent protein kinase (DNA-PK) is a cNHEJ factor that encompasses the Ku70-Ku80 (KU) heterodimer and the large catalytic subunit (DNA-PKcs). In mouse models, loss of DNA-PKcs (DNA-PKcs-/-) abrogates end-processing (e.g., hairpin-opening), but not end-ligation, while expression of the kinase-dead DNA-PKcs protein (DNA-PKcsKD/KD) abrogates end-ligation, suggesting a kinases-dependent structural function of DNA-PKcs during cNHEJ. Lymphocyte development is abolished in DNA-PKcs-/- and DNA-PKcsKD/KD mice due to the requirement for both hairpin-opening and end-ligation during V(D)J recombination. DNA-PKcs itself is the best-characterized substrate of DNA-PK. The S2056-cluster is the best characterized auto-phosphorylation site on human DNA-PKcs. Here we show that radiation can induce phosphorylation of murine DNA-PKcs at the corresponding S2053 and generated knockin mouse models with alanine- (DNA-PKcsPQR) or phospho-mimetic aspartate (DNA-PKcsSD) substitutions at the S2053 cluster. Despite moderate radiation sensitivity in the DNA-PKcsPQR/PQR fibroblasts and lymphocytes, both DNA-PKcsPQR/PQR and DNA-PKcsSD/SD mice retain normal kinase activity, and undergo efficient V(D)J recombination and class switch recombination, indicating that phosphorylation at the S2053-cluster of mouse DNA-PKcs (corresponding to S2056 of human DNA-PKcs), although important for radiation resistance, is dispensable for the end-ligation and hairpin-opening function of DNA-PK essential for lymphocyte development.
2019-04-17 | GSE129895 | GEO
Project description:The non-homologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of Kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters – PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But mice carrying alanine substitution at all 5 serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. XLF is a non-essential NHEJ factor. Xlf-/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, MRI, etc.) or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQRXlf-/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class switch recombination junctions from DNA-PKcsPQR/PQRXlf-/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation.
2023-05-01 | GSE228850 | GEO
Project description:GM0637 cell were pretreated with ATM inhibitor before adding DNA damaging agent neocarzinostatin (NCS), and cells were harvested after 4 hours for the microarray analyses of whole-genome miRNAs. To examine how miRNAs are regulated in the DNA damage response, we assessed the genome-wide mature miRNA expression in GM0637 cells treated with or without ATM inhibitor (ATM-IN)
2012-11-14 | E-GEOD-42248 | biostudies-arrayexpress
Project description:Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination
| PRJNA482753 | ENA
Project description:Analysis of gene expression changes in galactose-induced cataracts when an Atm inhibitors is added. A total of six samples were analyzed: a sample without induced turbidity (control), three samples in which turbidity was induced by galactose (galactose), and a sample with ATM inhibitor added (KU55933 or AZD0156).
2022-09-24 | GSE194074 | GEO