DNA-PKcs suppress chromosome translocation in human cells
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ABSTRACT:
PROVIDER: PRJNA974458 | ENA |
REPOSITORIES: ENA
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Project description:DNA-PKcs is a crucial component of the non-homologous end joining (NHEJ) repair machinery. To investigate its function in human cell lines, we conducted a study using K562 and HEK293T cell lines. We introduced twinned DNA double-strand breaks (DSBs) or genome-wide DSBs into these cell lines via nucleofection and transfection, respectively. Subsequently, we employed high-throughput genome translocation sequencing (HTGTS) to capture the translocation events (i.e., ligation between "prey(s)" and "bait") and the rejoining events (i.e., direct repair within the "bait" locus) under different conditions, including with or without DNA-PKcs inhibition or deletion. We quantified the number of translocation events by normalizing them to the number of rejoining events, denoted as TL. Interestingly, DNA-PKcs inhibition led to an increase in TL, indicating a higher frequency of translocations. However, it is important to note that chromosomal translocations still predominantly relied on NHEJ despite DNA-PKcs inhibition. Furthermore, we observed that DNA-PKcs deletion resulted in an elevated utilization of microhomology in translocation formation. Nevertheless, NHEJ remained the primary mechanism driving translocation events.These findings provide valuable insights into the role of DNA-PKcs in the repair pathways involved in translocation events in human cell lines. The utilization of HTGTS allowed us to comprehensively analyze the effects of DNA-PKcs inhibition and deletion, shedding light on the interplay between NHEJ and alternative repair mechanisms in translocation formation.
2023-09-01 | GSE232940 | GEO
Project description:Two DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous studies reported distinct repair mechanisms for chromosome translocation, with NHEJ predominantly involved in human and A-EJ in mice. NHEJ depends on DNA-PKcs, a critical partner in synapsis formation and downstream component activation. While DNA-PKcs inhibition promotes chromosome translocations harboring microhomologies in mice, its synonymous effect in human is not known. We find partial DNA-PKcs inhibition in human cell lines leads to increased genome-wide translocations composed mostly of direct joints, indicating the continued involvement of dampened NHEJ in these processes. In contrast, complete DNA-PKcs inhibition and genetic inhibition DNA-PKcs kinase domain substantially increased microhomology-mediated end joining (MMEJ), thus bridging the two different translocation mechanisms between human and mice. Similar to a previous study on Ku70 deletion, DNA-PKcs deletion in G1/G0-phase mouse pro-B cell lines, impair the recombination of RAG1/2-mediated DNA double-strand breaks (DSBs). This DNA-PKcs-deficient repair mechanism exhibited reduced V(D)J recombination efficiency, increased end resection, decreased polymerase-mediated insertions, loss of recombination fidelity and generated relatively higher rates of chromosome translocation as a consequence of dysregulated coding and signal end joining. Our study underscores DNA-PKcs in suppressing illegitimate chromosome rearrangement in both species.
2023-12-30 | GSE249453 | GEO
Project description:DNA-PKcs Suppresses Illegitimate Chromosome Rearrangements
| PRJNA1049124 | ENA
Project description:Long non-coding RNAs (lncRNAs) play important roles in cancer development and progression; however, their contributions to gastric cancer metastasis remain largely unknown. By lncRNA microarray screening, our study showed that 10 lncRNAs are dysregulated in gastric cancer tissues with or without lymph node metastasis, of which lnc-LEMGC ranks as one of the most significantly downregulated lncRNAs. Lnc-LEMGC inhibited cell migration and invasion both in vitro and in vivo, by combining with protein DNA-PKcs. Importantly, nucleotides 1,300–1,800 of lnc-LEMGC prevented DNA-PKcs phosphorylation of serine 2056 and partially abrogated the effects of downstream effectors, transforming growth factor alpha, EGFR, Src kinase, paxillin, and focal adhesion kinase, in the epidermal growth factor receptor (EGFR) pathway. The results of this study extend our knowledge of lncRNA’s molecular mechanisms, in which lnc-LEMGC functions by directly suppressing the phosphorylation of its combined protein DNA-PKcs and inactivating the DNA-PKcs downstream EGFR signaling.
2021-07-21 | GSE180443 | GEO
Project description:Long noncoding RNA lnc-LEMGC combines with DNA-PKcs to suppress gastric cancer metastasis
| PRJNA748266 | ENA
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 αNAC (alpha chain of the Nascent polypeptide-Associated Complex) transcriptional coregulator is developmentally expressed in osteoblasts and regulates osteoblast differentiation in vitro and in vivo. αNAC can activate or repress gene transcription, a function that is dynamically regulated by post-translational modification. Phosphorylation of residue Ser132 stimulates the sumoylation of αNAC on Lys127 to repress gene expression. Using in vitro kinase assays, we show that Ser132 phosphorylation is mediated by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Pharmacological inhibition of DNA-PKcs kinase activity or gene silencing of Prkdc (encoding DNA-PKcs) in murine osteoblastic MC3T3-E1 cells and human adipose-derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. ChIP-seq identified Ezh2 as a target of the αNAC/DNA-PKcs signaling pathway. Mechanistically, inhibition of DNA-PKcs repressed Ezh2 expression, induced cell cycle block, and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 (BMP-2) response in osteoblasts and other mesenchymal cells. Importantly, in vivo inhibition of the kinase enhanced bone biomechanical properties, and bones from osteoblast-specific conditional Prkdc-knockout mice exhibited increased stiffness. In conclusion, DNA-PKcs is a negative regulator of osteoblast differentiation, and therefore DNA-PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases.
2019-12-10 | GSE141686 | GEO
Project description:The DNA-dependent protein kinase (DNA-PK), composed of the KU heterodimer and the catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor1. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. Beyond DNA, KU also binds to RNA, with unknown significance in mammals. Using mouse models, we uncovered an unexpected role for DNA-PK in ribosomal RNA (rRNA) biogenesis and hematopoiesis. Expression of kinase-dead (KD) DNA-PKcs (DNA-PKcsKD/KD) abroagates cNHEJ2. But DNA-PKcsKD/KDTp53-/- mice develop myeloid disease rather than pro-B cell lymphoma, like other cNHEJ/Tp53-deficient mice3. DNA-PKcs is its own the best substrate. Blocking DNA-PKcs phosphorylation at the T2609, but not the S2056 cluster leads to KU-dependent 18S rRNA processing defects, compromises global protein synthesis in hematopoietic cells and causes bone marrow failure in mice. KU drives assembly of DNA-PKcs on a broad array of cellular RNAs, including the U3 small nucleolar RNA (snoRNA), which is essential for 18S rRNA processing4. U3 activates purified DNA-PK and triggers T2609 phosphorylation. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit (SSU) processome. Together our data show that DNA-PK has RNA-dependent, but cNHEJ-independent, functions during ribosome biogenesis that require DNA-PKcs’ kinase activity and T2609 cluster’s phosphorylation.
2020-01-05 | GSE109026 | GEO
Project description:<p>Kidney injury initiates epithelial dedifferentiation and myofibroblast activation during the progression of chronic kidney disease (CKD). Herein, we found that the expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was significantly increased in the kidney tissues of both CKD patients and CKD mice induced by unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion (UIR) injury. In vivo, knockout of DNA-PKcs or treatment with its specific inhibitor NU7441 hampered the development of CKD in mice. In vitro, DNA-PKcs deficiency preserved epithelial cell phenotype and inhibited fibroblast activation induced by transforming growth factor-beta 1 (TGF-beta-1). Additionally, our results showed that TBP-associated factor 7 (TAF7), as a possible substrate of DNA-PKcs, enhanced mTORC1 activation by upregulating RAPTOR expression, which subsequently promoted metabolic reprogramming in injured epithelial cells and myofibroblasts. Taken together, DNA-PKcs can be inhibited to correct metabolic reprogramming via the TAF7/mTORC1 signaling in CKD, and serve as a new target for treating CKD.</p>
2023-01-25 | MTBLS5971 | MetaboLights
Project description:The potential mechanisms of DNA-PKcs and its related signaling pathways in radiation-induced pulmonary toxicity is unclear. The current study utilized genetic engineering DNA-PKcs knockout mouse model, to investigate the molecular mechanisms after dose-response exposure of of the fractionated low-LET photon and high-LET carbon-ion exposure to the whole thorax.
2023-11-09 | E-MTAB-6419 | biostudies-arrayexpress