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Regulation of ALT-associated homology-directed repair by polyADP-ribosylation

Nature Structural & Molecular Biology volume 27, pages 1152–1164 (2020)Cite this article

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Abstract

The synthesis of poly(ADP-ribose) (PAR) reconfigures the local chromatin environment and recruits DNA-repair complexes to damaged chromatin. PAR degradation by poly(ADP-ribose) glycohydrolase (PARG) is essential for progression and completion of DNA repair. Here, we show that inhibition of PARG disrupts homology-directed repair (HDR) mechanisms that underpin alternative lengthening of telomeres (ALT). Proteomic analyses uncover a new role for poly(ADP-ribosyl)ation (PARylation) in regulating the chromatin-assembly factor HIRA in ALT cancer cells. We show that HIRA is enriched at telomeres during the G2 phase and is required for histone H3.3 deposition and telomere DNA synthesis. Depletion of HIRA elicits systemic death of ALT cancer cells that is mitigated by re-expression of ATRX, a protein that is frequently inactivated in ALT tumors. We propose that PARylation enables HIRA to fulfill its essential role in the adaptive response to ATRX deficiency that pervades ALT cancers.

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Fig. 1: Deregulation of PAR metabolism alters ALT activity.
Fig. 2: Disruption of PAR metabolism interferes with telomeric HDR.
Fig. 3: Identification of the PAR-regulated ALT proteome.
Fig. 4: Consequences of HIRA depletion on ALT activity.
Fig. 5: HIRA compensates for ATRX loss, and its depletion is synthetic lethal with ATRX loss.
Fig. 6: PAR-dependent regulation of HIRA-dependent chromatin assembly.

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Data availability

Original immunofluorescence and colony-formation images are separately available in the Figshare data depository (https://figshare.com/s/17f00c7faa765b329c22). Proteomics data has been deposited at ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020243. Source data are provided with this paper.

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Acknowledgements

We are indebted to M. Lund Nielsen for sharing detailed protocols and advice in relation to enrichment of PARylated proteins with AF1521. We are grateful to M. Altmeyer, D. Clynes, R. Greenberg, B. Johnson, K. Miller, S. Oesterreich, S. Polo, D. Slade, H. van Attikum and S. Smith for sharing high quality reagents. We thank G. Delgoffe, A. Menk and J. Stewart-Ornstein for guidance with experiments and sharing resources. Resources and facilities at the UPMC Hillman Cancer Center were supported by Comprehensive Cancer Center Support Grant NCI/no. P30CA047904. Research funding was provided to individual investigators from the following agencies; R.J.O. NCI/no. 5R01CA207209-02 and American Cancer Society no. RSG-18-038-01-DMC; S.C.W. NIH/no. 1S10OD019973-O1; A.I.N. NIH U24CA210967 and R01 GM094231; R.W.S. NIH/no. R01CA148629 and NIEHS/no. R01ES014811. N.K. is supported by a T32 training grant, NIGMS/no. T32GM008424-25. This work was also supported by grants C480/A11411 and C5759/A17098 from Cancer Research UK to D.I.J., I.D.W., K.M.S and D.O.; and ERC-2015-ADG- 694694 ‘ChromADICT’, ANR-16-CE15-0018 ‘CHRODYT’, ANR-16-CE11-0028 ‘REPLICAF’, ANR-16-CE12-0024 ‘CHIFT’, ITN-765966 Curie ‘EPISYSTEM’ and ITN-813327 Curie ‘CHROMDESIGN’ to G.A. and D.R.G.

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Authors and Affiliations

  1. Department of Pharmacology and Chemical Biology, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, USA

    Song My Hoang, Nicole Kaminski, Ragini Bhargava, Jonathan Barroso-González, Michelle L. Lynskey, Laura García-Expósito, Justin L. Roncaioli, Anne R. Wondisford & Roderick J. O’Sullivan

  2. Department of Cell Biology, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA, USA

    Callen T. Wallace & Simon C. Watkins

  3. Drug Discovery Unit, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, Macclesfield, UK

    Dominic I. James, Ian D. Waddell, Donald Ogilvie & Kate M. Smith

  4. Department of Pathology, University of Michigan, Ann Arbor, MI, USA

    Felipe da Veiga Leprevost, Dattatreya Mellacharevu & Alexey I. Nesvizhskii

  5. Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA

    Alexey I. Nesvizhskii

  6. Department of Pharmacology and the Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA

    Jianfeng Li & Robert W. Sobol

  7. Institut Curie, PSL Research University, CNRS, Sorbonne Université, Nuclear Dynamics Unit, Equipe Labellisée, Ligue contre le Cancer, Paris, France

    Dominique Ray-Gallet & Genevieve Almouzni

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  1. Song My Hoang
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Contributions

R.J.O. conceived and designed the study; S.M.H. designed and conducted all experimentation; R.J.O. and S.M.H analyzed the data. N.K., R.B., M.L.L., J.L.R., J.B.G., L.G.-E., A.R.W. and R.J.O. assisted with experimentation and analyzed data. C.T.W. and S.C.W. assisted with optical-imaging experiment design, optimization and analyzes. D.I.J., I.D.W., K.M.S. and D.O. produced, validated and provided the PARG inhibitors. F.V.L., D.M. and A.I.N. generated and analyzed proteomic data. J.L. and R.W.S. provided essential reagents. D.R.-G. and G.A. provided essential reagents, assisted with experimental design and interpretation of results. S.M.H. and R.J.O. wrote the manuscript with input from coauthors.

Corresponding author

Correspondence to Roderick J. O’Sullivan.

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Extended data

Extended Data Fig. 1 Disruption of PAR turnover perturbs recombinogenic activity at ALT telomeres.

a, Western blot of PARP1 or PARG knockdown and PAR accumulation in U2OS cells expressing shRNAs. Extracts from 2 mM hydroxyurea (HU)-PARGi (5μM/24hrs) treated U2OS cells serve as a control for PAR induction. b, Representative IF images of APBs (PML-TTAGGG) in U2OS cells expressing the indicated shRNAs. c, Quantification of APBs (% positive cells) in U2OS, Saos2 and HeLa LT cells expressing the indicated shRNAs. d, Quantification of telomere sister chromatid exchanges (t-SCE) (% per metaphase) in U2OS, Saos2 and HeLa LT cells expressing the indicated shRNAs. n refers to the number of metaphase spreads analyzed from N = 3 independent assays. e, Left: Western blot of U2OS cells transfected with non-targeting and/or RECQ1 siRNAs treated with PARPi (5μM/72hrs). Right: Quantification of t-SCEs in U2OS and HeLa LT cells transfected with NT siRNA, and/or RECQ1 siRNA. DMSO/PARPi (5μM/24hrs). n refers to the number of metaphase spreads analyzed from N = 4 independent assays. f, Cell cycle profiles and (g) cellular [ADP/ATP] ratio of U2OS and Hela LT cells treated with inhibitors for 12 days. h, PFGE of DMSO, PARPi (100 nM) or PARGi (1 μM) treated VA13 cells. The red dot indicates mean telomere length (kb). i, Representative images and quantification from clonogenic survival assays in ALT + cells, TEL + cells and (j) U2OS cells expressing ATRX treated with DMSO, PARPi (100μM) or PARGi (1μM) for 7 days. All scale bars in IF panels=5μm. All graphed data in the figure are mean ± s.e.m except d, e which are mean ± s.d. Unless otherwise stated, (n) is the number of cells analyzed and the number of independent assays (N) conducted is represented by black circles. Statistical significance was determined using one-way ANOVA except except (d) where the Mann-Whitney test was used and (i-j) where Students t test was used. Uncropped blots for a, e and digital images are deposited on Figshare. Graphed data is available as Source Data.

Source data

Extended Data Fig. 2 PARylation is an early and direct mediator of TRF1-FokI DSB formation.

a, [ADP/ATP] ratio in DMSO/PARGi treated WT-TRF1-FokI U2OS cells. Cells were treated with 1.5 mM/1 hr MMS. (b) Representative IF images and quantification of PAR at WT-TRF1-FokI DSBs after PARGi, PARGi-PARPi or TNKS1 knockdown. c, Representative IF images and quantification showing GFP-PARP1 localization in WT-TRF1-FokI cells treated with PARPi, PARGi or both. d, Representative IF images and quantification showing GFP-PARG localization in WT-TRF1-FokI U2OS cells. e, Left: Representative IF images and quantification of telomere foci size per cell in VA13 and Hela LT cells transfected with WT-TRF1-FokI from N = 2 independent assays. f, Representative stills of telomere (eGFP-TRF1) movement in U2OS cells treated with DMSO, PARPi or PARGi. Graph displays the cumulative Mean Squared Displacement (MSD) of 100 telomeres. g, Top: Schematic of DNA combing in G2-synchronized WT-TRF1-FokI cells treated with DMSO, PARPi, PARGi, or co-treated with PARPi and PARGi. Left: Quantification of telomeric fiber length of combined pulses. Right: Violin plot analysis of fork velocity. h, Graphs of CldU/IdU tract distribution of telomeric fibers in inhibitor treated U2OS-TRF1-FokI cells. n refers to the number of fibers containing TTAGGG signals analyzed from N = 2 independent assays. i, Representative IF images and quantification of BrdU synthesis at telomeres in the indicated cell lines after transfection with WT-TRF1-FokI and treated with PARGi or PARPi. j, Representative IF images and quantification of PCNA and (k) POLD3 localization at WT-TRF1-FokI telomeres treated with treated with DMSO, PARPi, PARGi, PARGi-Me or PARPi-PARGi. All inhibitor treatments, 5μM/4hrs unless otherwise indicated. All scale bars in IF panels=5μm. All graphed data in the figure are mean ± s.e.m. Unless otherwise stated, (n) is the number of cells analyzed and the number of independent assays (N) conducted is represented by black circles. Statistical significance was determined using one-way ANOVA. Digital images are deposited on Figshare. Graphed data is available as Source Data.

Source data

Extended Data Fig. 3 PAR-dependent recruitment of proteins to TRF1-FokI induced telomeric DSBs.

a, Spectral counts for the indicated proteins that were identified by Af1521-PAR proteomics. b, Western blot analysis illustrating the expression of GFP fusion proteins in U2OS WT-TRF1-FokI cells. GFP antibody was used to blot for protein expression in each treatment. c, Representative IF images showing the localization of the indicated GFP fusion proteins in WT-TRF1-FokI U2OS cells following treatment with DMSO, PARPi and combined PARGi/PARPi. d, In vivo PARylation assay with GFP tagged FUS, RBMX and ARP3. * indicates the band corresponding to the immunoprecipitated GFP-tagged target protein on PAR blots. e, Western blotting was performed with antibodies to validate siRNA knockdown of endogenous protein hits from Af1521-PAR proteomics in U2OS cells. f, Cell cycle profile of U2OS and VA13 cells after siRNA knockdown of protein hits from Af1521-PAR proteomics. All inhibitor treatments, 5μM/4hrs. All scale bars in IF panels=5μm. Uncropped blots for b, d-e and digital images are deposited on Figshare. Graphed data is available as Source Data.

Source data

Extended Data Fig. 4 Selectivity of HIRA for localization to telomeres in ALT cancer cells telomeric DSBs is independent of RPA and is necessary for telomere DNA synthesis.

a, Representative IF images of HIRA-YFP localization in ALT + and TEL + cell lines treated with DMSO/PARGi. b, Representative IF images of HIRA-YFP localization in U2OS cells after exposure to 30 J/m2ultra-violet C (UV-C) and 10 Gy ionizing irradiation (γIR). 5 μM PARGi was added for 30 mins following irradiation. c, Left: Western blot validation of RPA70 knockdown in U2OS cells. Middle: Representative IF images of HIRA-YFP localization at telomeres in U2OS cells after RPA70 knockdown. Right: Quantification of HIRA-YFP localization to telomeres in indicated conditions from N = 2 independent assays. d, Western blot validation of HIRA, CABIN1 and UBN1 siRNA knockdown in U2OS cells. e, Graphs of CldU/IdU tract distribution of >30 telomeric fibers in NT siRNA and HIRA siRNA transfected U2OS-TRF1-FokI cells. f, Representative IF images and quantification of BrdU synthesis at telomeres in indicated cell lines that are transfected with WT-TRF1-FokI and HIRA siRNA. All inhibitor treatments, 5 μM/4 hrs. All scale bars in IF panels=5 μm. Unless otherwise stated, (n) is the number of cells analyzed and the number of independent assays (N) conducted is represented by black circles. Uncropped blots for c-d and digital images are deposited on Figshare. Graphed data is available as Source Data.

Source data

Extended Data Fig. 5 HIRA compensates for and forms a synthetic lethal interaction with ATRX loss.

a, Representative IF images and quantification of HIRA-YFP localization in Hela LT cells transfected with ATRX siRNA and WT-TRF1-FokI, as well as treated with PARGi (5 μM, from N = 2 independent assays. b, Western blot validation of HIRA knockdown in ALT + and TEL + cell lines using two different shRNA sequences (#1 and #2). c, Western blot validation of HIRA and ATRX knockdown in TEL + HeLa LT cell lines using the indicated shRNAs. d, Representative images and quantification of clonogenic survival assays with Hela LT cell line stably expressing scrambled non-targeting (NT), ATRX (#A and #B), and HIRA (#1 and #2) shRNAs for 5 days. All inhibitor treatments, 5μM/4hrs. All scale bars in IF panels=5μm. Unless otherwise stated, (n) is the number of cells analyzed and the number of independent assays (N) conducted is represented by black circles. Uncropped blots for c-d and digital images are deposited on Figshare. Graphed data is available as Source Data.

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Extended Data Fig. 6 Analysis of potential binding of PAR by HIRA.

a, GFP-PARP1, YFP-HIRA or GFP-FUS were transiently transfected in U2OS cells. Immunoprecipitated and blotted GFP-fusion proteins were incubated with biotinylated PAR and detected using an anti-PAR (10H) antibody. GFP fusion proteins were detected with HRP conjugated GFP antibody. b, Western blot showing depletion of endogenous HIRA and complementation with the indicated HIRA constructs in U2OS cells. Uncropped blot images for panel a,b are shown in Supplementary Fig. 1.

Supplementary information

Supplementary Information

Supplementary Figure 1.

Reporting Summary

Supplementary Video 1

Telomere mobility in ALT U2OS cells treated with DMSO.

Supplementary Video 2

Telomere mobility in ALT U2OS cells treated with PARPi.

Supplementary Video 3

Telomere mobility in ALT U2OS cells treated with PARGi.

Supplementary Table 1

Listing of proteins identified by mass spectrometry.

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Hoang, S.M., Kaminski, N., Bhargava, R. et al. Regulation of ALT-associated homology-directed repair by polyADP-ribosylation. Nat Struct Mol Biol 27, 1152–1164 (2020). https://doi.org/10.1038/s41594-020-0512-7

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