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C1orf109L binding DHX9 promotes DNA damage depended on the R-loop accumulation and enhances camptothecin chemosensitivity.

ABSTRACT: Objectives: R-loop is a three-stranded nucleic acid structure of RNA/DNA hybrid, which occurs naturally during transcription, and more R-loop accumulation can trigger serious DNA damage. There has been increasing attention to the issue of R-loop accumulation acted as a target for cancer therapy. However, the regulation of R-loop-associated proteins is poorly explored.

Material and method: Quantitative real-time PCR and Western blot were used to measure the expression of C1orf109 in cell lines. In addition, C1orf109L (C1orf109 longest isoform) protein binding partner was identified and validated using immunoprecipitation-mass spectrometric (IP-MS) and immunoprecipitation assays. DNA-RNA immunoprecipitation (DR-IP) and immunofluorescence determined the C1orf109L location on R-loop. R-loop accumulation was determined by immunofluorescence. Cell cycle was determined by flow cytometry. Finally, time-lapse assay and cell counting were conducted to determined cell survival in response to camptothecin (CPT).

Results: We found that C1orf109L could mediate cell cycle arrest in the G2/M phase and DNA damage depended on R-loop accumulation. Meanwhile, C1orf109L could bind with DHX9 to trigger R-loop accumulation. And C1orf109L was competitive with PARP1 binding to DHX9, which would block the function of DHX9-PARP1 to prevent the R-loop accumulation. Furthermore, C1orf109L could enhance the chemosensitivity of CPT, a chemotherapeutic drug capable of promoting R-loop formation.

Conclusions: Our data demonstrate that C1orf109L triggers R-loop accumulation and DNA damage to arrest cell cycle.

SUBMITTER: Dou P

PROVIDER: S-EPMC7507383 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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C1orf109L binding DHX9 promotes DNA damage depended on the R-loop accumulation and enhances camptothecin chemosensitivity.

Dou Peng P Li Yiqun Y Sun Haoxiu H Xie Wanqiu W Zhang Xiaoqing X Zhang Xiaohan X Zhang Dandan D Qiao Shupei S Ci Yanpeng Y Nie Huan H Han Fang F Li Yu Y

Cell proliferation 20200806 9

<h4>Objectives</h4>R-loop is a three-stranded nucleic acid structure of RNA/DNA hybrid, which occurs naturally during transcription, and more R-loop accumulation can trigger serious DNA damage. There has been increasing attention to the issue of R-loop accumulation acted as a target for cancer therapy. However, the regulation of R-loop-associated proteins is poorly explored.<h4>Material and method</h4>Quantitative real-time PCR and Western blot were used to measure the expression of C1orf109 in ...[more]

PMID: 32761833

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Project description:BackgroundNF-κB signaling is widely linked to the pathogenesis and treatment resistance in cancers. Increasing attention has been paid to its anti-oncogenic roles, due to its key functions in cellular senescence and the senescence-associated secretory phenotype (SASP). Therefore, thoroughly understanding the function and regulation of NF-κB in cancers is necessary prior to the application of NF-κB inhibitors.MethodsWe established glioblastoma (GBM) cell lines expressing ectopic TCF4N, an isoform of the β-catenin interacting transcription factor TCF7L2, and evaluated its functions in GBM tumorigenesis and chemotherapy in vitro and in vivo. In p65 knock-out or phosphorylation mimic (S536D) cell lines, the dual role and correlation of TCF4N and NF-κB signaling in promoting tumorigenesis and chemosensitivity was investigated by in vitro and in vivo functional experiments. RNA-seq and computational analysis, immunoprecipitation and ubiquitination assay, minigene splicing assay and luciferase reporter assay were performed to identify the underlying mechanism of positive feedback regulation loop between TCF4N and the p65 subunit of NF-κB. A eukaryotic cell-penetrating peptide targeting TCF4N, 4N, was used to confirm the therapeutic significance.ResultsOur results indicated that p65 subunit phosphorylation at Ser 536 (S536) and nuclear accumulation was a promising prognostic marker for GBM, and endowed the dual functions of NF-κB in promoting tumorigenesis and chemosensitivity. p65 S536 phosphorylation and nuclear stability in GBM was regulated by TCF4N. TCF4N bound p65, induced p65 phosphorylation and nuclear translocation, inhibited its ubiquitination/degradation, and subsequently promoted NF-κB activity. p65 S536 phosphorylation was essential for TCF4N-led senescence-independent SASP, GBM tumorigenesis, tumor stem-like cell differentiation and chemosensitivity. Activation of p65 was closely connected to alterative splicing of TCF4N, a likely positive feedback regulation loop between TCF4N and p65 in GBM. 4N increased chemosensitivity, highlighting a novel anti-cancer strategy.ConclusionOur study defined key roles of TCF4N as a novel regulator of NF-κB through mutual regulation with p65 and provided a new avenue for GBM inhibition.

Dataset Information

C1orf109L binding DHX9 promotes DNA damage depended on the R-loop accumulation and enhances camptothecin chemosensitivity.

Publications

C1orf109L binding DHX9 promotes DNA damage depended on the R-loop accumulation and enhances camptothecin chemosensitivity.

Similar Datasets

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