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Extrachromosomal telomere repeat DNA is linked to ALT development via cGAS-STING DNA sensing pathway

Nature Structural & Molecular Biology volume 24, pages 1124–1131 (2017)Cite this article

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Abstract

Extrachromosomal telomere repeat (ECTR) DNA is unique to cancer cells that maintain telomeres through the alternative lengthening of telomeres (ALT) pathway, but the role of ECTRs in ALT development remains elusive. We found that induction of ECTRs in normal human fibroblasts activated the cGAS-STING-TBK1-IRF3 signaling axis to trigger IFNβ production and a type I interferon response, resulting in cell-proliferation defects. In contrast, ALT cancer cells are commonly defective in sensing cytosolic DNA. We found that STING expression was inhibited in ALT cancer cell lines and transformed ALT cells. Notably, the ALT suppressors histone H3.3 and the ATRX–Daxx histone chaperone complex were also required to activate the DNA-sensing pathway. Collectively, our data suggest that the loss of the cGAS-STING pathway may be required to evade ECTR-induced anti-proliferation effects and permit ALT development, and this requirement may be exploited for treatments specific to cancers utilizing the ALT pathway.

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Figure 1: Defective DNA sensing in ALT cell lines.
Figure 2: ECTR production elicits IFN responses in human fibroblasts.
Figure 3: ECTR activates cGAS-STING-dependent innate immune signaling for IFNβ induction.
Figure 4: ECTRs cause cGAS- and type I IFN-dependent proliferation defects.
Figure 5: Los s of STING expression in ALT cell lines.
Figure 6: ATRX, Daxx and H3.3 mediate activation of the DNA-sensing pathway.

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Acknowledgements

We thank R.R. Reddel (Children's Medical Research Institute) for providing cell line samples; M.-C. Yao, R.-H.Chen, M.-Z. Lai, C. Wen and J. Lingner for discussions; H.-C. S. Yen for the cGAS construct; T.E. Chen for technical help; and the Genomics Core, Bioinformatics-Biology Core and Imaging Core in the Institute of Molecular Biology at Academia Sinica for help with data collection and analysis. Research in the laboratory of L.-Y. Chen was supported by Career Development Award CDA-105-L01 from Academia Sinica and grants from the Ministry of Science and Technology (105-2311-B-001-055-MY3).

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Authors and Affiliations

  1. Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

    Yi-An Chen, Yi-Ling Shen, Hsuan-Yu Hsia, Yee-Peng Tiang, Tzu-Ling Sung & Liuh-Yow Chen

  2. Taiwan International Graduate Program in Molecular and Cellular Biology, Academia Sinica and Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan

    Yi-An Chen & Liuh-Yow Chen

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Y.-A.C. and L.-Y.C. designed the study. Y.-A.C. performed most of the experiments, with assistance from Y.-L.S., H.-Y.H. and Y.-P.T. Y.A.C., T.-L.S. and L.-Y.C. wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Analyses of ECTRs by C-circle and T-circle assays.

(a) C-circle and (b) T-circle amplification reactions using genomic DNA from different cell lines in the presence (+) or absence (-) of phi29 DNA polymerase. Using 32P-labeled telomeric probes, C-circle reaction products and 50ng of denatured genomic DNA (input) were analyzed by dot blots, and T-circle reaction products were detected by in-gel hybridization. Asterisks denote ALT cell lines.

Supplementary Figure 2 ECTRs in U2OS cells do not induce IFNβ expression.

(a-b) Dot blots analysis of extrachromosomal DNA (ecDNA) in U2OS cells prepared by fractionation using 32P-labeled telomeric and Alu probes. Quantifications of radioisotope signals were shown in b. Singles of native (Nat.) and denatured (Den.) samples indicate single-stranded DNA and total DNA, respectively. (c) Immunofluorescence staining with anti-cGAS antibody (green) coupled with Tel-FISH (red) in U2OS cells expressing flag-cGASm2A. Nuclei were visualized by DAPI staining (blue). Enlarged images show co-localization between cytoplasmic ECTRs and cGAS. Scale bar, 10 μm. Quantifications of ECTR positive cells (≥ 2 cytoplasmic telomeric foci) and co-localization between ECTRs and cGAS are shown (right). The ECTR positive cells with and without co-localization between cytoplasmic ECTRs and cGAS are indicated as +cGAS and -cGAS, respectively. The n values indicate the numbers of cells analyzed. (d) T-circle assay of U2OS/Vector and U2OS/ERT2-TRF2∆B cells. The cells treated with or without 4-OHT for 48 h were subjected to genomic DNA extraction and subsequent T-circle amplification reactions using phi29 DNA polymerase. Linear telomere restriction fragments (TRFs) and phi29-dependent amplification products of TCs were detected by in-gel hybridization using 32P-labeled telomeric probes. (e) Real-time RT-PCR analysis of IFNβ mRNA in U2OS/Vector and U2OS/ERT2-TRF2∆B cells treated with 4-OHT for 48 h. U2OS/Vector cells transfected with poly(I:C) served as a positive control. (mean ± s.d.; n=3 technical replicates of representatives of three independent experiments) n.s., not significant, ***P≤0.001, paired t-test.

Supplementary Figure 3 Analysis of IFNβ expression in BJTL cells expressing ERT2-TRF2 and ERT2-TRF2ΔB.

(a) Western blots and (b) immunofluorescence staining using anti-myc antibody in BJTL cells stably expressing vector, myc-ERT2-TRF2 or myc-ERT2-TRF2ΔB and treated with 4-OHT for the indicated times. Telomeric foci and nuclei were visualized following Tel-FISH and DAPI staining, respectively. Scale bar, 10 μm. Arrows in b indicate representative telomeric localization of myc-ERT2-TRF2 and ERT2-TRF2ΔB. (c) Analysis of IFNβ-mRNA by real-time RT-PCR in the cells in a treated with 4-OHT for 48 h. (d-f) Comparison between transient and prolong 4-OHT treatments in BJTL/ERT2-TRF2ΔB cells. Schematic of experimental design of 4-OHT treatments was shown in d. Tel-FISH (red) and DAPI staining (blue) of 4-OHT-untreated (-) and -treated (+) BJTL/ERT2-TRF2ΔB cells were shown in e. Scale bar, 10 μm. Arrows indicate cytoplasmic telomeric foci. IFNβ-mRNA levels were analyzed by real-time RT-PCR (f).

Supplementary Figure 4 ECTRs induce expression of IFNβ and ISGs and affect cell proliferation in cGAS and STING dependent manner.

(a) Real-time RT-PCR analysis of mRNA levels of indicated genes in BJTL/ERT2-TRF2ΔB cells treated with/without 4-OHT for 48 h. Error bars indicate +/- s.e.m. of triplicate technical replicates and are representative of three independent experiments (n.s., not significant, *P≤0.05, **P≤0.01, paired t-test). (b) ELISA analyses of secreted IFNβ and CXCL10 from BJTL/ERT2-TRF2ΔB cells transfected with control (siCtrl: non-target) or cGAS siRNAs for 48 h, followed by 4-OHT treatments for 96 h. Error bars indicate +/- s.e.m. of triplicate technical replicates and are representative of three independent experiments (n.d., not detectable, *P≤0.05, **P≤0.01, unpaired t-test). (c-d) Western blot analyses of BJTL/ERT2-TRF2ΔB cells transfected with control (siCtrl: non-target), cGAS or STING siRNAs for 48 h using antibodies against cGAS, STING and GAPDH. (e-f) Real-time RT-PCR analysis of IFNβ mRNA in BJTL/ERT2-TRF2∆B cells transfected with control (siCtrl: non-target), cGAS or STING siRNAs for 48 h, followed by 4-OHT treatment for 48 h. (g-h) Cell growth analyses of BJTL/ERT2-TRF2ΔB cells transfected with indicated siRNAs for 48 h followed by 4-OHT treatment for 36 h. Cell numbers were analyzed at day 4 after 4-OHT addition. Relative growth rate indicates the cell number ratio of +4-OHT to -4-OHT samples. (mean ± s.d.; n=3 technical replicates of representatives of three independent experiments) n.s., not significant, *P≤0.05, **P≤0.01, ***P≤0.01, paired t-test.

Supplementary Figure 5 Silencing cGAS and p53 rescue cell proliferation.

(a) Western blot analyses of BJTL/ERT2-TRF2ΔB cells transfected with control (siCtrl: non-target), cGAS or p53 (TP53) siRNAs for 48 h using antibodies against cGAS, TP53, p21 and GAPDH. p21 expression is transcriptionally regulated by p53, so depletion of p53 reduces p21 protein levels. (b) Cell growth analyses of BJTL/ERT2-TRF2ΔB cells transfected with siRNAs for 48 h, followed by 4-OHT treatment for 36 h. Cell numbers were analyzed at day 4 after 4-OHT addition. Relative growth rate indicates the cell number ratio of +4-OHT to -4-OHT samples. (mean ± s.d.; n=3 independent experiments) n.s., not significant, **P≤0.01, ***P≤0.001, paired t-test.

Supplementary Figure 6 Functional and expression analyses of STING in ALT cell lines.

(a) Functional assay of STING using cGAMP. Cells were permeabilized with digitonin and treated with cGAMP (100 nM) for 4 h, followed by real-time RT-PCR analyses of IFNβ mRNA. (b) Relative STING mRNA levels of BJTL and in vitro-transformed ALT cell lines. (mean ± s.d.; n=3 technical replicates of representatives of three independent experiments)

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Chen, YA., Shen, YL., Hsia, HY. et al. Extrachromosomal telomere repeat DNA is linked to ALT development via cGAS-STING DNA sensing pathway. Nat Struct Mol Biol 24, 1124–1131 (2017). https://doi.org/10.1038/nsmb.3498

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