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RTEL1 suppresses G-quadruplex-associated R-loops at difficult-to-replicate loci in the human genome

Nature Structural & Molecular Biology volume 27, pages 424–437 (2020)Cite this article

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Abstract

Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as ‘MiDAS’. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.

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Fig. 1: RTEL1 is essential for MiDAS following replication stress.
Fig. 2: RTEL1 is essential for the maintenance of genome stability.
Fig. 3: RTEL1 prevents R-loop accumulation in interphase cells.
Fig. 4: DRIP-seq analysis reveals that sequences enriched in RTEL1-depleted or APH-treated cells share similar features.
Fig. 5: RTEL1 depletion induces an accumulation of G4s, which inhibits cell growth.
Fig. 6: MEF cells expressing ATPase-dead (K48R) Rtel1 have increased R-loops in interphase and reduced MiDAS in prometaphase.
Fig. 7: RTEL1 acts downstream of SLX4 in the MiDAS pathway.
Fig. 8: Overexpression of RNase H1 can remove R-loops, and partially blocks MiDAS under RS conditions.

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Data availability

The raw sequence data for each DRIP-seq sample are deposited at NCBI Bioproject (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA510030/). The names of the samples are YL_sample_7.fq.gz (siRNA-control), YL_sample_8.fq.gz (siRNA-control+RNase H), YL_sample_9.fq.gz (siRNA-RTEL1), YL_sample_10.fq.gz (siRNA-RTEL1+RNase H), YL_sample_11.fq.gz (siRNA-control+APH) and YL_sample_12.fq.gz (siRNA-control+APH+RNase H). The final processed data are deposited at the NCBI GEO database (accession no. GSE129907). The names of the samples are: sample_7.bed (siRNA-control), sample_8.bed (siRNA-control+RNase H), sample_9.bed (siRNA-RTEL1), sample_10.bed (siRNA-RTEL1+RNase H), sample_11.bed (siRNA-control+APH) and sample_12.bed (siRNA-control+APH+RNase H). The source data for graphs, western blots and DNA gels are available with the paper online. The source data for all of the images in figures are deposited at Figshare (https://doi.org/10.6084/m9.figshare.11833368).

Code availability

Custom-made Bash and Python scripts are available upon request from the corresponding authors.

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Acknowledgements

We thank members of the Hickson and Liu groups for helpful discussions, and J.P. Diaz and E. Hoffmann for critical reading of the manuscript. We also thank W. Qiao and V. Lee (BGI Hong Kong) for processing the DRIP-seq samples. This work was supported by grants from the Chinese Scholarship Council (PhD fellowship 201406170048; W.W.), the Danish Medical Research Council (Postdoc fellowship DFF-4004-00155B; R.B.), the European Commission (FP7 Marie Curie Fellowship; Ö.Ö.), US NIH grants (GM56888 and U54 OD020355; J.H.P.), US NCI MSK Cancer Center core grant P30 CA008748 (J.H.P.), The Nordea Foundation of Denmark (I.D.H.), The Danish National Research Foundation (DNRF115; I.D.H. and Y.L.) and the European Commission (H2020/ Marie Skłodowska-Curie, 859853; I.D.H. and Y.L.).

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Author notes

  1. Özgün Özer

    Present address: Institute of Cancer Research, London, UK

  2. Liqun Ren

    Present address: The Basic Medical Research Institute, Chengde Medical University, Chengde, China

  3. These authors contributed equally: Wei Wu, Rahul Bhowmick.

Authors and Affiliations

  1. Center for Chromosome Stability, University of Copenhagen, Copenhagen, Denmark

    Wei Wu, Rahul Bhowmick, Ivan Vogel, Özgün Özer, Roshan S. Thakur, Philipp H. Richter, Liqun Ren, Ian D. Hickson & Ying Liu

  2. Laboratory of Chromosome Biology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

    Fiorella Ghisays, Esther Sanchez de Leon & John H. Petrini

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Contributions

W.W., R.B., Ö.Ö., F.G., R.S.T., E.S., P.H.R. and L.R. carried out experiments. I.V. performed analysis of bioinformatic data. W.W., R.B., J.H.P., I.D.H. and Y.L. designed experiments and interpreted results. I.D.H. and Y.L. wrote the manuscript and all authors edited it.

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Correspondence to Ian D. Hickson or Ying Liu.

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Extended data

Extended Data Fig. 1 RTEL1 depletion by 3’UTR siRNAs causes R-loop accumulation in interphase and reduced MiDAS in prometaphase cells under replication stress conditions.

a, Experimental workflow for the analysis of MiDAS in prometaphase cells in U2OS cells following RTEL1 depletion and APH treatment. b & c, Representative images of Immunoblotting analysis (b) and quantification (c) of RTEL1 expression following transfection with a control and two 3’UTR targeting siRNA. GAPDH was used as a loading control. The quantification of relative protein levels was performed using Fiji/ImageJ software. Error bars indicate mean ± s.d. (N = 3). **P < 0.01, based on two-tailed Student’s t-test. d & e, Representative IF images of three independent experiments (d) and quantification (e) of interphase cells treated with control siRNA or RTEL1 3’UTR siRNA for 48 hours and stained with S9.6 antibody (red) and FANCD2 antibody (green). A zoomed image for the area indicated by a white box is shown on the right. Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. f & g, Representative IF images (f) and quantification (g) of MiDAS foci (labeled with EdU; red) in prometaphase cells treated as shown in panel a. DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. Scale bar is 10 µm. Data for graphs in panels c,e,g are available as Source data. Uncropped image for panel b is available as Source data.

Source data

Extended Data Fig. 2 RTEL1 is essential for MiDAS in telomerase positive cells under replication stress conditions.

a, Experimental workflow for the analysis of MiDAS in prometaphase HeLa or HCT116 cells following RTEL1 depletion and APH treatment. b & c, Representative images (b) and quantification (c) of RTEL1 expression following siRNA transfection with a control and RTEL1 siRNAs. Tubulin was used as a loading control. The quantification of relative protein levels was performed using Fiji/ImageJ software. Error bars indicate mean ± s.d. (N = 3). ****P < 0.0001, based on two-tailed Student’s t-test d & e, Representative IF images (d) and quantification (e) of MiDAS (indicated with EdU; red) in prometaphase cells. DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. Scale bar is 10 µm. Data for graphs in panels c,e are available as Source data. Uncropped image for panel b is available as Source data.

Source data

Extended Data Fig. 3 Following RTEL1 depletion, R-loops are accumulated in telomerase positive cells.

a, Experimental workflow for the analysis of R-loops in interphase HeLa or HCT116 cells following RTEL1 depletion. b & c, Representative IF images (b) and quantification (c) of interphase HeLa cells stained with S9.6 antibody specific for R-loops (red), or a nucleolin antibody (green). DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. d & e, Representative IF images (d) and quantification (e) of interphase HCT116 cells stained with S9.6 antibody specific for R-loops (red), or a nucleolin antibody (green). DNA was stained with DAPI (blue). The S9.6 signal intensity per nucleus was calculated by Fiji/ImageJ software and determined by subtracting the S9.6 staining with that from nucleoli (defined by nucleolin; green) in each nucleus. In each case, a specificity control for S9.6 staining is shown, in which fixed cells were treated with RNase H to eliminate R-loops. Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. Scale bar is 10 µm. Data for graphs in panels c,e are available as Source data.

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Extended Data Fig. 4 R-loops accumulate at specific regions of the genome following RTEL1 depletion.

a, Experimental workflow of cell synchronization and siRNA treatment for analyzing G1 phase U2OS cells. b, Representative IF images of G1 cells transfected with control siRNA or RTEL1 siRNA and stained with S9.6 antibody specific for R-loops (green) or a 53BP1 antibody (red). DNA was stained with DAPI (blue). Scale bar is 10 µm. c, Quantification of co-localization between 53BP1 bodies and R-loops in G1 cells from panel b. Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. d, Experimental workflow for the quantitative analysis of R-loops enriched at different loci of the genome following RTEL1 depletion by DNA-RNA immunoprecipitation (DRIP) qPCR. e, Quantification of R-loops by DRIP qPCR at different genomic loci. The qPCR value was normalized against a region of the ZNF554 gene for each sample. Error bars indicate mean ± s.d. *p < 0.05; **p < 0.01, based on two-tailed Student’s t-test. (N for each locus: NRG3 = 8, FRA3B = 7, FRA16D = 4, FRA7B = 7, FRA10C = 3, 18 S = 5, 28 S = 3). Data for graphs in panels c,e are available as Source data.

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Extended Data Fig. 5 RTEL1 depletion induces chromosome fragility at common fragile sites.

a, Experimental workflow for analyzing chromosome fragility in U2OS cells. b, Representative images of FISH signal on chromosomes from cells treated with either an siRNA control (siCon), an siRNA targeting RTEL1 (siRTEL1), or 0.4 μM APH. n, the number of loci examined. Loci of FRA16D (green) or FRA3B (red) were detected by FISH probes. DNA was stained with DAPI. c, Quantification of chromosome fragility at FRA16D or FRA3B. Error bars indicate mean ± s.d. (N = 3). *p < 0.05; **p < 0.01, based on two-tailed Student’s t-test. Scale bar is 2 µm. Data for graphs in panel c is available as Source data.

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Extended Data Fig. 6 The analysis of un-bound protein and cell cycle profiles at different stages of interphase or mitosis following RTEL1 depletion and APH (0.4 µM) treatment.

The un-bound proteins was assessed by western blot analysis (a,c,e). GAPDH or Tubulin was used as loading controls. Histone H3 was used as a loading control for the chromatin-bound fraction. The cell cycle profiles (b,d,f) were output from Flowjo software and representative for two independent experiments. Uncropped images for panel a,c,e are available as Source data.

Extended Data Fig. 7 SLX4 depletion reduces the frequency of MiDAS in a U2OS cell line with inducible GFP-SLX4.

a, Experimental workflow of analyzing SLX4 in interphase cells or MiDAS in prometaphase cells by IF following SLX4 depletion. b, Representative images of a U2OS cell line with expression of GFP-SLX4 following the treatment with doxycycline (+Dox) to induce expression. GFP-SLX4 (green) or SLX4 (red) was detected by direct fluorescence (green; for GFP) or using antibodies against SLX4 respectively. c & d, Representative images of MiDAS (labeled with EdU; red) (c) and quantification (d) in prometaphase U2OS cells following SLX4 depletion by a 3’UTR siRNA or with inducible GFP-SLX4 expression. DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. Scale bar is 10 µm. Data for graphs in panel d is available as Source data.

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Extended Data Fig. 8 SETX does not play a role in MiDAS.

a, Experimental workflow for analyzing prometaphase U2OS cells by IF following SETX depletion and APH treatment. b, Representative western blotting analysis of SETX following siRNA treatment. Actin was used as a loading control. c, d, Representative images of MiDAS (labeled with EdU; red) (c) and quantification (d) in prometaphase U2OS cells following SETX depletion. DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ns: p > 0.05, based on two-tailed non-parametric Mann Whitney test. Data for graphs in panel d is available as Source data. Uncropped image for panel b is available as Source data.

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Extended Data Fig. 9 RNase H1 does not play a role in MiDAS.

a, Experimental workflow of analyzing interphase or prometaphase U2OS cells by IF following APH treatment and RNase H1 depletion alone or combined with RTEL1 depletion. b & c, Representative IF images (b) and quantification (c) of interphase cells stained with S9.6 antibody (red) and FANCD2 antibody (green). A zoomed image for the area indicated by a white box is shown on the right. Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. d, Western blotting analysis of RNase H1 or RTEL1 following siRNA treatment. Actin was used as a loading control. e & f, Representative images of MiDAS (labeled with EdU; red) (e) and quantification (f) in prometaphase cells following RNaseH1 depletion alone or combined with RTEL1 depletion. Scale bar, 10 μm.DNA was stained with DAPI (blue). Error bars represent median ± s.e.m. (N = 3). ****p < 0.0001, based on two-tailed non-parametric Mann Whitney test. Data for graphs in panels c,f are available as Source data. Uncropped image for panel d is available as Source data.

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Supplementary information

Supplementary Information

A cover page, reagents table, note, Figs. 1–3 and legends.

Reporting Summary

Supplementary Table 1

An Excel file of the peaks identified in DRIP-seq sample 7 (siRNA-control) and sample 9 (siRNA-RTEL1), excluding the peaks not affected by RNase H treatment. A P value was assigned to each peak, to assess whether it was differentially enriched in sample 9 against sample 7. In addition, these peaks are annotated with their genomic location and other features, including CFSs, G4 motifs, transcribed genes, COSMIC cancer mutations, COSMIC cancer gene expression and promoters.

Supplementary Table 2

An Excel file of the peaks identified in DRIP-seq sample 7 (siRNA-control) and sample 11 (siRNA-control+APH), excluding the peaks not affected by RNaseH treatment. A P value was assigned to each peak to assess whether it was differentially enriched in sample 11 against sample 7. In addition, these peaks have been annotated with their genomic location and other features, including CFSs, G4 motifs, transcribed genes, COSMIC cancer mutations, COSMIC cancer gene expression and promoters.

Supplementary Table 3

An Excel file of all of the CFSs annotated by three published databases in 2010, 2012 and 2019, respectively (see Supplementary Note).

Supplementary Data

Source data for unprocessed Western Blots and gels.

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Wu, W., Bhowmick, R., Vogel, I. et al. RTEL1 suppresses G-quadruplex-associated R-loops at difficult-to-replicate loci in the human genome. Nat Struct Mol Biol 27, 424–437 (2020). https://doi.org/10.1038/s41594-020-0408-6

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