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GUARDIN is a p53-responsive long non-coding RNA that is essential for genomic stability

Nature Cell Biology volume 20, pages 492–502 (2018)Cite this article

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Abstract

The list of long non-coding RNAs (lncRNAs) involved in the p53 pathway of the DNA damage response is rapidly expanding, but whether lncRNAs have a role in maintaining the de novo structure of DNA is unknown. Here, we demonstrate that the p53-responsive lncRNA GUARDIN is important for maintaining genomic integrity under steady-state conditions and after exposure to exogenous genotoxic stress. GUARDIN is necessary for preventing chromosome end-to-end fusion through maintaining the expression of telomeric repeat-binding factor 2 (TRF2) by sequestering microRNA-23a. Moreover, GUARDIN also sustains breast cancer 1 (BRCA1) stability by acting as an RNA scaffold to facilitate the heterodimerization of BRCA1 and BRCA1-associated RING domain protein 1 (BARD1). As such, GUARDIN silencing triggered apoptosis and senescence, enhanced cytotoxicity of additional genotoxic stress and inhibited cancer xenograft growth. Thus, GUARDIN may constitute a target for cancer treatment.

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Fig. 1: Identification of GUARDIN as a p53-inducible lncRNA.
Fig. 2: GUARDIN is expressed at higher levels in colon cancers with wild-type p53 than in those carrying mutant p53.
Fig. 3: GUARDIN is important for cell survival and proliferation.
Fig. 4: GUARDIN sequesters miR-23a to stabilize TRF2.
Fig. 5: GUARDIN binds to and stabilizes BRCA1.
Fig. 6: GUARDIN protects genomic integrity through TRF2 and BRCA1.
Fig. 7: GUARDIN protects cells from apoptosis induced by genotoxic insults through TRF2 and BRCA1.

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Acknowledgements

We thank X. Xu of Shenzhen University for providing U2OS-HR and U2OS-NHEJ cells and T. Ohta of St. Marianna University for providing the HA-BARD1 plasmid. We thank L. Kong and Q. Cheng of Henan Provincial People’s Hospital for their assistance with preparation of tissue sections and immunohistochemistry experiments. This work was supported by grants from the National Key R&D Program of China (2016YFC1302302) and the National Natural Science Foundation of China (81430065, 31371388, 31601117 and 81471551).

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  1. These authors contributed equally: Wang Lai Hu, Lei Jin, An Xu.

Authors and Affiliations

  1. Chinese Academy of Sciences (CAS) Key Laboratory of Innate Immunity and Chronic Disease, CAS Centre for Excellence in Cell and Molecular Biology, Innovation Centre for Cell Signalling Network, School of Life Sciences, University of Science and Technology of China, Hefei, China

    Wang Lai Hu, An Xu & Mian Wu

  2. Translational Research Institute, Henan Provincial People’s Hospital, Zhengzhou, China

    Wang Lai Hu, Rick F. Thorne, Xu Dong Zhang & Mian Wu

  3. Department of Immunology, Anhui Medical University, Hefei, China

    Wang Lai Hu

  4. School of Medicine and Public Health, University of Newcastle, Callaghan, New South Wales, Australia

    Lei Jin & Xu Dong Zhang

  5. Department of Pathophysiology, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China

    Yu Fang Wang

  6. School of Environmental and Life Sciences, University of Newcastle, Newcastle, New South Wales, Australia

    Rick F. Thorne

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Contributions

W.L.H., L.J., A.X., X.D.Z. and M.W. designed the research. W.L.H., L.J. and A.X. performed most of the experiments and data analysis. Y.F.W. participated in the experiments and data analysis. R.F.T. participated in the data analysis and manuscript preparation. M.W., R.F.T. and X.D.Z. wrote the manuscript.

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Correspondence to Xu Dong Zhang or Mian Wu.

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Integrated supplementary information

Supplementary Figure 1 lncRNA#6, GUARDIN, is regulated by wildtype p53 and its depletion induces apoptosis and reduced cell viability.

(a) p53-upregulated lncRNAs, 1, 2, 6, 7 and 8 identified by lncRNA profiling along with lincRNA-p21 and DINO were validated after induction of p53 in H1299 cells carrying an inducible wild-type p53 expression system using qPCR. Induced expression of p53 is shown as inset. Mean ± s.e.m.; n=3 independent experiments, two-tailed Student’s t-test. (b) Silencing of lncRNA#6 but not 1, 2, 7 or 8 triggered activation of caspase-3 and cleavage of PARP in HCT116 cells. Data shown represent three independent experiments. (c) Silencing of lncRNA#6 but not 1, 2, 7 or 8 caused reduction in cell viability in HCT116 cells. Mean ± s.e.m.; n=3 independent experiments, two-tailed Student’s t-test. (d) shRNA knockdown efficacy of lncRNA#1, 2, 6, 7 and 8 in HCT116 cells. Mean ± s.e.m.; n=3 independent experiments, two-tailed Student’s t-test. (e) The longest isoform of GUARDIN, RP3-510D11.2-1, was markedly more abundant than the others (RP3-510D11.2-2 and RP3-510D11.2-3) in H1299 cells with or without induction of p53. Mean ± s.e.m.; n=3 independent experiments, two-tailed Student’s t-test. (f) Ectopic expression wild-type p53 but not p53 mutants, p53R175H and p53R273H caused upregulation of GUARDIN in H1299 cells. Mean ± s.e.m.; n=3 independent experiments, two-tailed Student’s t-test. Statistics source data for a and c-f are provided in Supplementary Table 7. Uncropped images of blots for a, b and f in Supplementary Fig. 7.

Supplementary Figure 2 GUARDIN expression in colon cancer cells and tissues and the relation between miR-34a and MIR34AHG expression, copy number loss and p53 status.

(a) Schematic illustration of the genomic location of GUARDIN at chromosome 1p36.22 and its relation to the genes encoding miR-34a, MIR34AHG, and H6PD. (b) GUARDIN expression was positively correlated with the expression of miR-34a and MIR34AHG in laser capture micro-dissected (LCM) cancer cells from fresh surgical samples of 40 colon cancers. Linear regression analysis. (c) GUARDIN expression along with the expression of miR-34a and MIR34AHG were reduced in LCM-dissected colon cancer cells with copy number loss of their genes. n=40 biologically independent samples, two-tailed Student’s t-test. (d) Silencing or overexpression of GUARDIN did not alter the expression of miR-34a and its target Snail as well as MIR34AHG. n=3 independent experiments, two-tailed Student’s t-test. (e) Introduction of anti-miR-23a or knockdown of MIR34AHG did not affect expression of GUARDIN. n=3 independent experiments, two-tailed Student’s t-test. (f) Schematic illustrations of the putative p53-binding region (p53-BR) located 305-329 bp upstream of the GUARDIN transcriptional start site and the pGL3-basic based GUARDIN promoter reporter constructs. (g) In situ hybridization analysis of GUARDIN expression in HCT116 cells and HCT116 cells with GUARDIN knocked down by shRNA that were used as positive and negative biological controls, respectively. Data shown represent three independent experiments. (h) GUARDIN expression in LCM-dissected colon cancer cells from tumours without copy number loss of its gene, similar to the expression of p21 mRNA, was lower in mutant TP53 cases compared with paired LCM pre-neoplastic epithelial cells (hyperplastic polyps (n=5 biologically independent samples) and adenomas (n=7 biologically independent samples)), whereas there was no significant difference in GUARDIN and p21 mRNA expression between LCM-dissected pre-neoplastic and paired normal colon epithelial cells. n=12 biologically independent samples. two-tailed Student’s t-test. Statistics source data for b-e and h are provided in Supplementary Table 7.

Supplementary Figure 3 Rescue of the inhibitory effects of GUARDIN depletion by pan-caspase inhibitor z-VAD-fmk and the identification of complementarity between GUARDIN and miR-23a.

(a) Knockdown of GUARDIN in HCT116 cells using independent shRNAs. Mean ± s.e.m.; n=3 independent experiments, two-tailed Student’s t-test. (b) Treatment with z-VAD-fmk abolished activation of caspase-3 and cleavage of PARP caused by silencing of GUARDIN in HCT116 cells. Data shown represent three independent experiments. (c) Treatment with z-VAD-fmk partially reversed inhibition of cell number expansion caused by silencing of GUARDIN in HCT116, U2OS, and A549 cells. Mean ± s.e.m.; n=3 independent experiments, two-tailed Student’s t-test. (d) Schematic Illustration of base pairing between miR-23a and GUARDIN. (e) Schematic illustration of psiCHECK2-based luciferase reporter constructs containing wild-type GUARDIN (psiCHECK2-GUARDIN-WT) and a mutant reporter construct in which three putative miR-23a binding sites were mutated (psiCHECK2-GUARDIN-mt). Mutated bases are depicted in red. Statistics source data for a and c are provided in Supplementary Table 7. Uncropped images of blots for b are shown in Supplementary Fig. 7.

Supplementary Figure 4 EMSA assays confirm the GUARDIN-BRCA1-BARD1 interaction with domain mapping experiments identifying distinct regions of GUARDIN which bind to BRCA1 and BARD1.

(a) GUARDIN physically associates with BRCA1 and BARD1 in electrophoretic mobility-shift assays. Data shown represent three independent experiments. (b) Schematic illustration of division of GUARDIN into three fragments corresponding to individual exons of the GUARDIN gene (E1, E2 and E3) along with corresponding deletion mutants used. (c) The fragment of GUARDIN corresponding to exon 3 of its gene was required for its association with BARD1. Data shown represent three independent experiments. (d) The fragments of GUARDIN corresponding to exon 1 and 2 of its gene were necessary for its association with BRCA1. Data shown represent three independent experiments. Uncropped images of blots for a, c and d are shown in Supplementary Fig. 7.

Supplementary Figure 5 GUARDIN binding to BRCA1 and BARD1 occurs through their respective amino-terminal domains but independently of embodied RING domains.

(a) Schematic illustration of BRCA1 and the corresponding mutants with deletion of individual fragments. FL: Full length; NT: N Terminus; SCD: serine cluster domain; CT: C Terminus; ΔRING: RING domain deletion. (b) Schematic illustration of BARD1 and the corresponding mutants with deletion of individual fragments. FL: Full length; NT: N Terminus; FAR: Four ankyrin repeats; CT: C Terminus; ΔRING: RING domain deletion. (c) RNA immunoprecipitation assays were performed in HCT116 cells transfected with indicated BRCA1 segment cDNA. The NT of BRCA1 was required for its association with endogenous GUARDIN. Data shown represent three independent experiments. (d) RNA immunoprecipitation assays were performed in HCT116 cells transfected with the indicated BARD1 construct. The NT of BARD1 was required for its association with endogenous GUARDIN. Data shown represent three independent experiments. (e) RNA immunoprecipitation assays were performed in HCT116 cells transfected with indicated BRCA1 segment cDNA. The RING domain encompassed within the NT of BRCA1 was not required for its association with endogenous GUARDIN. Data shown represent three independent experiments. (f) RNA immunoprecipitation assays were performed in HCT116 cells transfected with indicated BARD1 segment cDNA. The RING domain encompassed within the NT of BARD1 was not required for its association with endogenous GUARDIN. Data shown represent three independent experiments. Uncropped images of blots for c-f are shown in Supplementary Fig. 7.

Supplementary Figure 6 GUARDIN depletion induces cell cycle arrest and defective DNA repair through effects on BRCA1, TRF2 and/or miR-23a.

(a) Silencing of GUARDIN caused cell cycle arrest in the G0/G1 phase in HCT116 cells. Mean ± s.e.m.; n=3 independent experiments, two-tailed Student’s t-test. (b) Treatment with z-VAD-fmk did not affect reduction in activation of the HR and NHEJ repair pathways caused by silencing of GUARDIN. Mean ± s.e.m.; n=3 independent experiments, two-tailed Student’s t-test. (c) Neither overexpression of TRF2, introduction of anti-miR-23a, nor overexpression of BRCA1 alone significantly affected inhibition of DDR activation caused by GUARDIN knockdown, whereas co-overexpression of TRF2 and BRCA1 or co-introduction of anti-miR-23a and BRCA1-expressing constructs abolished reduction in DDR activation in cells with GUARDIN knocked down. Mean ± s.e.m.; n=3 independent experiments, two-tailed Student’s t-test. (d) Overexpression of TRF2, introduction of anti-miR-23a, or overexpression of BRCA1 alone did not rescue HCT116 cells from GUARDIN knockdown, whereas the combination of TRF2 overexpression or anti-miR-23a and BRCA1 overexpression abolished inhibition of cell viability caused by GUARDIN knockdown. Mean ± s.e.m.; n=3 independent experiments, two-tailed Student’s t-test. (e) Model depicting the GUARDIN-mediated pathway required to maintain genomic stability. Statistics source data for a-d are provided in Supplementary Table 7.

Supplementary Figure 7 Uncropped images from Western blots.

Uncropped images for Fig.1a-j. Uncropped images for Fig. 3f and Fig. 4g, i-k. Uncropped images for Fig. 5b-j. Uncropped images for Fig. 7b, c, Supplementary Fig. 1a, b, f, and Supplementary Fig. 3b. Uncropped images for Supplementary Fig. 4a, c, d and Supplementary Fig. 5c-f.

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Hu, W.L., Jin, L., Xu, A. et al. GUARDIN is a p53-responsive long non-coding RNA that is essential for genomic stability. Nat Cell Biol 20, 492–502 (2018). https://doi.org/10.1038/s41556-018-0066-7

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  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-018-0066-7

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