Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination-deficient cells

Nature Cell Biology volume 23, pages 160–171 (2021)Cite this article

Subjects

Abstract

The response to poly(ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair and the abundance of lesions that trap PARP enzymes. It remains unclear, however, if the established role of PARP in promoting chromatin accessibility impacts viability in these settings. Using a CRISPR-based screen, we identified the PAR-binding chromatin remodeller ALC1/CHD1L as a key determinant of PARPi toxicity in HR-deficient cells. ALC1 loss reduced viability of breast cancer gene (BRCA)-mutant cells and enhanced sensitivity to PARPi by up to 250-fold, while overcoming several resistance mechanisms. ALC1 deficiency reduced chromatin accessibility concomitant with a decrease in the association of base damage repair factors. This resulted in an accumulation of replication-associated DNA damage, increased PARP trapping and a reliance on HR. These findings establish PAR-dependent chromatin remodelling as a mechanistically distinct aspect of PARPi responses and therapeutic target in HR-deficient cancers.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Loss of ALC1 reduces proliferation and confers olaparib hypersensitivity in BRCA-mutant cells.
Fig. 2: ALC1 loss causes PARPi hypersensitivity in HR-deficient cells.
Fig. 3: ALC1 loss mitigates PARPi resistance in BRCA-mutant cells that are deficient in PARP1 or PARG.
Fig. 4: Loss of ALC1 increases genomic instability.
Fig. 5: ALC1 function in base damage repair is not epistatic with HR and single-strand break repair.
Fig. 6: ALC1 function in the DNA damage response requires PARP1 and PARP2.
Fig. 7: ALC1 PAR-recognition and chromatin-remodelling activities are essential for responses to PARPi and MMS.
Fig. 8: Cooperation between ALC1 with PARP activity facilitates chromatin-directed DNA repair.

Similar content being viewed by others

Data availability

Sequencing data generated in this study have been deposited in the Gene Expression Omnibus with accession codes GSE149104 (for RNA-seq) and GSE150955 (for ATAC-seq). Data from the CRISPR screen have been provided as mapped reads in Supplementary Table 1. Functionally conserved domains were identified using either the NCBI Conserved Domain Search or UniProt. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

All the analyses were based on standard algorithms described in the Methods and referenced accordingly. There are no custom algorithms to make available.

References

  1. Langelier, M.-F., Planck, J. L., Roy, S. & Pascal, J. M. Structural basis for DNA damage-dependent poly(ADP-ribosyl)ation by human PARP-1. Science 336, 728–732 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Langelier, M.-F., Riccio, A. A. & Pascal, J. M. PARP-2 and PARP-3 are selectively activated by 5′ phosphorylated DNA breaks through an allosteric regulatory mechanism shared with PARP-1. Nucleic Acids Res. 42, 7762–7775 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gupte, R., Liu, Z. & Kraus, W. L. PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes. Genes Dev. 31, 101–126 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C. & Mandel, P. Poly(ADP-ribosyl)ation of polynucleosomes causes relaxation of chromatin structure. Proc. Natl Acad. Sci. USA 79, 3423–3427 (1982).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Krishnakumar, R. et al. Reciprocal binding of PARP-1 and histone H1 at promoters specifies transcriptional outcomes. Science 319, 819–821 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Hakmé, A., Wong, H.-K., Dantzer, F. & Schreiber, V. The expanding field of poly(ADP-ribosyl)ation reactions. ‘Protein modifications: beyond the usual suspects’ review series. EMBO Rep. 9, 1094–1100 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Pascal, J. M. The comings and goings of PARP-1 in response to DNA damage. DNA Repair 71, 177–182 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Luijsterburg, M. S. et al. PARP1 Links CHD2-mediated chromatin expansion and H3.3 deposition to DNA repair by non-homologous end-joining. Mol. Cell 61, 547–562 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Mateos-Gomez, P. A. et al. Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination. Nature 518, 254–257 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ronson, G. E. et al. PARP1 and PARP2 stabilise replication forks at base excision repair intermediates through Fbh1-dependent Rad51 regulation. Nat. Commun. 9, 746 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Ray Chaudhuri, A. et al. Topoisomerase I poisoning results in PARP-mediated replication fork reversal. Nat. Struct. Mol. Biol. 19, 417–423 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Fisher, A. E. O., Hochegger, H., Takeda, S. & Caldecott, K. W. Poly(ADP-ribose) polymerase 1 accelerates single-strand break repair in concert with poly(ADP-ribose) glycohydrolase. Mol. Cell. Biol. 27, 5597–5605 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Pines, A. et al. PARP1 promotes nucleotide excision repair through DDB2 stabilization and recruitment of ALC1. J. Cell Biol. 199, 235–249 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bryant, H. E. et al. PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination. EMBO J. 28, 2601–2615 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Golia, B., Singh, H. R. & Timinszky, G. Poly-ADP-ribosylation signaling during DNA damage repair. Front. Biosci. 20, 440–457 (2015).

    Article  CAS  Google Scholar 

  16. Bryant, H. E. et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913–917 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917–921 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Helleday, T. The underlying mechanism for the PARP and BRCA synthetic lethality: clearing up the misunderstandings. Mol. Oncol. 5, 387–393 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Murai, J. et al. Trapping of PARP1 and PARP2 by clinical PARP inhibitors. Cancer Res. 72, 5588–5599 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gogola, E., Rottenberg, S. & Jonkers, J. Resistance to PARP inhibitors: lessons from preclinical models of BRCA-associated cancer. Annu. Rev. Cancer Biol. 3, 235–254 (2019).

    Article  Google Scholar 

  21. Shi, J. et al. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nat. Biotechnol. 33, 661–667 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Najm, F. J. et al. Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens. Nat. Biotechnol. 36, 179–189 (2018).

    Article  CAS  PubMed  Google Scholar 

  23. Menear, K. A. et al. 4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one: a novel bioavailable inhibitor ofpoly(ADP-ribose) polymerase-1. J.Med. Chem. 51, 6581–6591 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Horton, J. K. et al. XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks. Cell Res. 18, 48–63 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Lord, C. J. & Ashworth, A. BRCAness revisited. Nat. Rev. Cancer 16, 110–120 (2016).

    Article  CAS  PubMed  Google Scholar 

  26. Xu, G. et al. REV7 counteracts DNA double-strand break resection and affects PARP inhibition. Nature 521, 541–544 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bunting, S. F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Jaspers, J. E. et al. Loss of 53BP1 causes PARP inhibitor resistance in Brca1-mutated mouse mammary tumors. Cancer Discov. 3, 68–81 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Wang, Y. et al. The BRCA1-Δ11q alternative splice isoform bypasses germline mutations and promotes therapeutic resistance to PARP inhibition and cisplatin. Cancer Res. 76, 2778–2790 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Pettitt, S. J. et al. Genome-wide and high-density CRISPR-Cas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance. Nat. Commun. 9, 1849 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Michelena, J. et al. Analysis of PARP inhibitor toxicity by multidimensional fluorescence microscopy reveals mechanisms of sensitivity and resistance. Nat. Commun. 9, 2678 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Gogola, E. et al. Selective loss of PARG restores PARylation and counteracts PARP inhibitor-mediated synthetic lethality. Cancer Cell 35, 950–952 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ceccaldi, R. et al. Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair. Nature 518, 258–262 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tsuda, M. et al. ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair. PLoS ONE 12, e0188320 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Ooka, M. et al. Chromatin remodeler ALC1 prevents replication-fork collapse by slowing fork progression. PLoS ONE 13, e0192421 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Ahel, D. et al. Poly(ADP-ribose)-dependent regulation of DNA repair by the chromatin remodeling enzyme ALC1. Science 325, 1240–1243 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ensminger, M. et al. DNA breaks and chromosomal aberrations arise when replication meets base excision repair. J. Cell Biol. 206, 29–43 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Caldecott, K. W. Single-strand break repair and genetic disease. Nat. Rev. Genet. 9, 619–631 (2008).

    Article  CAS  PubMed  Google Scholar 

  39. Lehmann, A. R. & Fuchs, R. P. Gaps and forks in DNA replication: rediscovering old models. DNA Repair 5, 1495–1498 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Gottschalk, A. J. et al. Poly(ADP-ribosyl)ation directs recruitment and activation of an ATP-dependent chromatin remodeler. Proc. Natl Acad. Sci. USA 106, 13770–13774 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lehmann, L. C. et al. Mechanistic insights into autoinhibition of the oncogenic chromatin remodeler ALC1. Mol. Cell 68, 847–859 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Singh, H. R. et al. A Poly-ADP-ribose trigger releases the auto-inhibition of a chromatin remodeling oncogene. Mol. Cell 68, 860–871 (2017).

    Article  CAS  PubMed  Google Scholar 

  43. Hanzlikova, H. et al. The importance of poly(ADP-ribose) polymerase as a sensor of unligated Okazaki fragments during DNA replication. Mol. Cell 71, 319–331 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hanzlikova, H., Gittens, W., Krejcikova, K., Zeng, Z. & Caldecott, K. W. Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin. Nucleic Acids Res. 45, 2546–2557 (2017).

    CAS  PubMed  Google Scholar 

  45. Chittori, S., Hong, J., Bai, Y. & Subramaniam, S. Structure of the primed state of the ATPase domain of chromatin remodeling factor ISWI bound to the nucleosome. Nucleic Acids Res. 47, 9400–9409 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Karras, G. I. et al. The macro domain is an ADP-ribose binding module. EMBO J. 24, 1911–1920 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Sellou, H. et al. The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage. Mol. Biol. Cell 27, 3791–3799 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Smith, R., Sellou, H., Chapuis, C., Huet, S. & Timinszky, G. CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation. Nucleic Acids Res. 46, 6087–6098 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Smith, R. et al. Poly (ADP-ribose)-dependent chromatin unfolding facilitates the association of DNA-binding proteins with DNA at sites of damage. Nucleic Acids Res. 47, 11250–11267 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Tarsounas, M. & Sung, P. The antitumorigenic roles of BRCA1–BARD1 in DNA repair and replication. Nat. Rev. Mol. Cell Biol. 21, 284–299 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Wong, R. P., García-Rodríguez, N., Zilio, N., Hanulová, M. & Ulrich, H. D. Processing of DNA polymerase-blocking lesions during genome replication is spatially and temporally segregated from replication forks. Mol. Cell 77, 3–16.e4 (2020).

    Article  CAS  PubMed  Google Scholar 

  52. Cong, K. et al. PARPi synthetic lethality derives from replication-associated single-stranded DNA gaps. Preprint at bioRxiv https://doi.org/10.1101/781989 (2019).

  53. Lopes, M., Foiani, M. & Sogo, J. M. Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions. Mol. Cell 21, 15–27 (2006).

    Article  CAS  PubMed  Google Scholar 

  54. Quinet, A. et al. PRIMPOL-mediated adaptive response suppresses replication fork reversal in BRCA-deficient cells. Mol. Cell 77, 461–474 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Branzei, D. & Szakal, B. Building up and breaking down: mechanisms controlling recombination during replication. Crit. Rev. Biochem. Mol. Biol. 52, 381–394 (2017).

    Article  CAS  PubMed  Google Scholar 

  56. Marians, K. J. Lesion bypass and the reactivation of stalled replication forks. Annu. Rev. Biochem. 87, 217–238 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Nagaraju, G. & Scully, R. Minding the gap: the underground functions of BRCA1 and BRCA2 at stalled replication forks. DNA Repair 6, 1018–1031 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Dungrawala, H. et al. The replication checkpoint prevents two types of fork collapse without regulating replisome stability. Mol. Cell 59, 998–1010 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Liu, X. et al. ERCC6L2 promotes DNA orientation-specific recombination in mammalian cells. Cell Res. 30, 732–744 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Zimmermann, M. et al. CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions. Nature 559, 285–289 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. DeWeirdt, P. C. et al. Genetic screens in isogenic mammalian cell lines without single cell cloning. Nat. Commun. 11, 752 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Gier, R. A. et al. High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening. Nat. Commun. 11, 3455 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Verma, P. et al. RAD52 and SLX4 act nonepistatically to ensure telomere stability during alternative telomere lengthening. Genes Dev. 33, 221–235 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Di Veroli, G. Y. et al. Combenefit: an interactive platform for the analysis and visualization of drug combinations. Bioinformatics 32, 2866–2868 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Gatti, M., Imhof, R., Huang, Q., Baudis, M. & Altmeyer, M. The ubiquitin ligase TRIP12 limits PARP1 trapping and constrains PARP inhibitor efficiency. Cell Rep. 32, 107985 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Quinet, A., Carvajal-Maldonado, D., Lemacon, D. & Vindigni, A. DNA fiber analysis: mind the gap! Methods Enzymol. 591, 55–82 (2017).

    Article  CAS  PubMed  Google Scholar 

  68. Petrovic, J. et al. Oncogenic notch promotes long-range regulatory interactions within hyperconnected 3D cliques. Mol. Cell 73, 1174–1190 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank D. Durocher (University of Toronto, Lunenfeld) for sharing hTERT-RPE1 p53−/− BRCA1−/− cells and controls, J. and R. Connaway (Stowers) for ALC1 plasmids, K. Caldecott (University of Sussex) for sharing the GFP-XRCC1 plasmid and hTERT-RPE1 XRCC1−/− and parental control cells, N. Lakin (University of Oxford) for sharing U-2 OS PARP1−/−, PARP2−/− and PARP1/2−/− cells, N. Johnson (Fox Chase) for SUM149PT BRCA1 reversion mutant cell lines and helpful discussions on mouse xenograft experiments and M. Altmeyer (University of Zurich) for helpful suggestions on PARP1 trapping experiments. This work was supported by NIH grants GM101149 and CA17494 (R.A.G.), who is also supported by funds from the Penn Center for Genome Integrity, the Basser Center for BRCA, a V Foundation Team Convergence Award and a Gray Foundation Team Science Award. P.V. was supported by the Ann and Sol Schreiber Mentored Investigator Award (Ovarian Cancer Research Fund Alliance) and pilot funds from the Ovarian Cancer Translational Center for Excellence (UPenn). J.S. was supported by the Michele and Kevah Konner Award through the Basser Center for BRCA.

Author information

Authors and Affiliations

  1. Department of Cancer Biology, Penn Center for Genome Integrity, Basser Center for BRCA, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Priyanka Verma, Peter V. Deraska, Moniher Deb, Weihua Li, Yue Shao, Yiwen Li, Sonali Patankar & Roger A. Greenberg

  2. Departments of Pathology and Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Yeqiao Zhou & Robert B. Faryabi

  3. Department of Cancer Biology, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Zhendong Cao, Eri Arai & Junwei Shi

  4. Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Laura Puentes & Robert H. Mach

Authors
  1. Priyanka Verma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yeqiao Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhendong Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter V. Deraska
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Moniher Deb
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eri Arai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Weihua Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yue Shao
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Laura Puentes
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yiwen Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Sonali Patankar
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Robert H. Mach
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Robert B. Faryabi
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Junwei Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Roger A. Greenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.V., J.S. and R.A.G. designed the study. P.V. did most of the experiments, with assistance from P.V.D., M.D., Y.S., Y.L. and S.P. Z.C. and E.A. performed the CRISPR screen in SUM149PT and CAPAN-1 cells and the RNA-seq experiments. Y.Z. carried out the ATAC-seq experiment under the guidance of R.B.F. W.L. performed the mouse work. L.P. imaged and analysed PARP1 and PARP2 trapping under the guidance of R.H.M. P.V. and R.A.G. wrote the manuscript with contributions from J.S.

Corresponding authors

Correspondence to Junwei Shi or Roger A. Greenberg.

Ethics declarations

Competing interests

R.A.G. is a founder and scientific advisory board member of RADD Pharmaceuticals. The other authors declare no competing interests.

Additional information

Peer review information Nature Cell Biology thanks Matthias Altmeyer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 ALC1 loss renders olaparib hypersensitivity and proliferation defects in various BRCA-mutant lines.

a, Protein domains ranked on the basis of the CRISPR score (CS) for ola sensitivity in BRCA1-mutant UWB1.289 cells. b, Immunoblot showing depletion of ALC1 using a sgRNA vector with GFP selection marker. GFP-positive cells were sorted and analyzed. The blot is a representative image of two biologically independent experiments. c, GFP competition assay to examine the effects of ALC1 depletion on ola sensitivity in CAPAN-1, SUM149PT and UWB1.289 cells. Ola sensitivity in CAPAN-1 and SUM149PT was confirmed using six and seven independent guides respectively and data for each guide are from one experiment performed at three different ola concentrations. Ola sensitivity in UWB1.289 was confirmed using seven independent guides and data for each guide is mean of two biologically independent experiments in the absence and presence of 50 nM ola concentration. Source data are provided.

Source data

Extended Data Fig. 2 ALC1 loss enhances the therapeutic window of olaparib sensitivity in BRCA-mutant cells.

a, Sensitivities of the indicated cell lines to ola using CellTiter-Glo. Data are mean of 2 (hTERT-RPE1) or mean ± s.e.m. of 3 (UWB1.289, SUM149PT and DLD1) biologically independent experiments. b, Representative images (left) and quantification (right) of the clonogenic survival of ALC1-depleted DLD1 WT and BRCA2-mutant cells grown in the presence of increasing concentrations of ola. Data are mean ± s.e.m. from 3 biologically independent experiments. Source data are provided.

Source data

Extended Data Fig. 3 Extended analysis of PARPi sensitivity upon ALC1 loss.

a, Sensitivities of the indicated DLD1 BRCA2-/- cells to vel (veliparib), ola and tal in CellTiter-Glo assay. Data are mean ± s.e.m. from n = 3 biologically independent experiments. b, Immunoblot showing ALC1 levels in cells used for xenograft studies (first four left lanes) and in tumors that reached >10.5 mm in any dimension, which is when the mice were euthanized. Data from two biologically independent tumors per condition are shown. c, GFP competition experiment in UWB1.289+BRCA1 addback line to examine the effects of the combined loss of ALC1 and the indicated DNA repair proteins on cell proliferation and ola sensitivity. Data are normalized to Tinitial and indicate mean ± s.e.m. After every two population doublings, cells were passaged (P) and GFP percent was recorded (n=4 independent transductions except for sgFEN1, where n = 6 independent transductions were performed). Source data are provided.

Source data

Extended Data Fig. 4 Genomic lesions in PARPi treated ALC1-deficient cells are repaired by SSBR and NHEJ.

a-b, Immunoblot showing levels of ALC1 and XRCC1 in indicated DLD1 (a) and hTERT-RPE1 (b) cells. The western samples were analyzed once to check the efficiency of the sgRNAs for protein depletion before drug sensitivity assays. c, Sensitivities of the indicated DLD1 cells lines to ola and tal using the CellTiter-Glo assay. Data are mean ± s.e.m. from n = 3 biologically independent experiments. d, Sensitivities of the indicated hTERT-RPE1 cells lines to tal using the CellTiter-Glo assay. Data are mean from n=2 biologically independent experiments. e, Sensitivities of the indicated UWB1.289 cell lines to ola using the CellTiter-Glo assay. Data are mean ± s.e.m. from n = 3 biologically independent experiments. f, Quantification of γH2AX-Rad51 foci in indicated cell lines. Cells were fixed 16 hrs. after treatment with 10 Gy ionizing radiation (IR). Median is indicated. p-value determined by Mann–Whitney was derived from n≥54 cells examined over two biologically independent experiments. g, Quantification of γH2AX-Rad51 foci in indicated cell lines. Cells were treated with 5 µM ola for 24 hrs. before fixation. Median is indicated. p-value determined by Mann–Whitney was derived from n≥114 cells examined over three biologically independent experiments. h, Representative images and quantification of radials (indicated by red arrowheads) and breaks (indicated by yellow arrowheads) in the indicated UWB1.289 cell lines, post treatment with 1 µM ola for 24 hrs. For each experiment, at least 50 spreads were analyzed per sample. Data are mean from two biologically independent experiments. Source data are provided.

Source data

Extended Data Fig. 5 ALC1 deficiency results in increased trapping of PARP1 and PARP2 by PARPi upon DNA damage.

a-b Representative images (left) and quantification (right) of PARP1(a) and PARP2 (b) trapping in UWB1.289 cells. Indicated treatments were performed for 4 hours. Median is indicated. p-value determined by Mann–Whitney was derived from n≥107 cells sampled over two biologically independent experiments.

Source data

Extended Data Fig. 6 ALC1 loss confers MMS sensitivity and results in replication-coupled gaps.

a, Representative images of Rad51-γH2AX foci in U-2 OS (left) and UWB1.289+BRCA1 (right) cell lines Scale bar: 10 microns. Images represent n≥67 cells examined over two biologically independent experiments. b, Representative images of γH2AX signal in EdU negative (left) and positive (right) hTERT-RPE1 BRCA1-/- cells. Scale bar: 10 microns. Images represent n≥99 cells examined over two biologically independent experiments. c, Sensitivities of the indicated cells lines to MMS and CPT using the CellTiter-Glo assay. Data are mean ± s.e.m. from n=3 biologically independent experiments. d, Sensitivities of the indicated hTERT-RPE1 cells lines to MMS and CPT using the CellTiter-Glo assay. Data are mean ± s.e.m. from n = three biologically independent experiments. e, Representative images of fibers from the S1 nuclease experiment. Scale bar: 2 microns. Images represent n≥75 fibers examined over two biologically independent experiments. Source data are provided.

Source data

Extended Data Fig. 7 ALC1 is recruited to the damaged chromatin under conditions of reduced PARylation.

a, Schematic of the experiment. b-c, Representative images (b) and quantification (c) of HA-ALC1 localization to chromatin upon indicated treatments in U-2 OS cells. d, Schematic of the experiment. e-f, Representative images (e) and quantification (f) of HA-ALC1 localization to chromatin upon indicated treatments in SUM149PT cells. Scale bar, 10 microns. The median value was normalized to untreated control. Data are mean ± s.e.m. from n = three biologically independent experiments, p-value, unpaired Student’s t-test. For each experiment, at least 50 cells were analyzed per sample. Source data are provided.

Source data

Extended Data Fig. 8 ATPase activity, H4 interaction and macrodomains of ALC1 are essential for protecting BRCA-mutant cells from ola hypersensitivity.

a, Representative images (left) and quantification (right) of the clonogenic survival assay using hTERT-RPE1 BRCA1-/- cells expressing sgALC1 and the indicated ALC1 mutants treated with ola (1 nM). Data are mean from two biologically independent experiments. b, Representative images (left) and quantification (right) of the clonogenic survival assay (left) using SUM149PT cells expressing sgALC1 and ALC1 K77R mutant treated with ola (0.5 nM). Data are mean ± s.e.m from n = three independent experiments. Number of colonies in the ola treated condition were normalized to its respective untreated counterpart. c, Sequence alignment of various chromatin remodelers using Clustal Omega. Histone H4 interacting residues as predicted by PDB:6PWF are highlighted and marked by a blue star. d, Immunoblots showing interactions of FLAG ALC1 WT and FLAG ALC1 D377A+ D381A with histone H4. Experiment was repeated twice with similar outcomes. e, Representative images of the clonogenic survival assay (left) and quantification (right) of SUM149PT cells expressing sgALC1 and indicated ALC1 macrodomain mutants treated with ola (1 nM). Data are mean ± s.e.m. from n = three independent experiments. Number of colonies in the ola treated condition were normalized to its respective untreated counterpart. Source data are provided.

Source data

Extended Data Fig. 9 ALC1 co-operates with PARP activity to permit association of repair proteins with chromatin.

a, DLD1 BRCA2–/– cells were fractionated and the chromatin-bound proteins were immunoblotted. Cells were treated with 5 µM ola for 4 hrs. Data for DLD1 BRCA2–/– cells are from the same sample, from two different western blots and the histone levels for each blot are shown by the ponceau staining. The data for XRCC1 is representative of 5 biologically independent experiments and the data for NTHL1 and APE1 is representative of 3 biologically independent experiments. b, Immunoblot of whole cell lysates of DLD1 BRCA2-/- cells showing total proteins levels upon ALC1 depletion and PARPi treatment. Cells were treated with indicated PARPi for 4 hrs. Data is representative of two biologically independent experiments. c, UWB1.289 cells were fractionated and the chromatin-bound proteins were immunoblotted. Cells were treated with 1 µM tal for 4 hrs. Data for XRCC1 is representative of 4 biologically independent experiments and the data for APE1 is representative of 3 biologically independent experiments. d, Immunoblot showing expression levels of HA-XRCC1. The blot was performed once to access the expression level of the tagged protein. e, Schematic of the IF experiment. f-g, Representative images (f) and quantification (g) of HA-XRCC1 localization to chromatin upon indicated treatments. Scale bar, 50 microns. Data are mean ± s.e.m. from n = three biologically independent experiments, p-value, unpaired Student’s t-test. For each cell line, the median value upon MMS treatment was normalized to its respective untreated control. Source data are provided.

Source data

Extended Data Fig. 10 ALC1 loss synergistically enhances IR sensitivity at low olaparib doses.

a-b Representative images of two independent clonogenic survival assay to monitor the effect of combining low doses of ola and IR upon ALC1 depletion in UWB1.289 (a) and hTERT-RPE1 BRCA1-/- cells (b). c-d, Quantification of clonogenic survival (c) and heat map of bliss scores (d) obtained from BRCA1–/– hTERT-RPE1 cells treated with the indicated doses of ola and IR. Data are mean from two biologically independent experiments. Bliss score >0, synergistic; Bliss score <0, antagonistic; Bliss score = 0, additive. Number of colonies in IR-treated conditions were normalized to their respective un-irradiated counterparts. Colonies with more than 50 cells were included in the analysis. Source data are provided.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

Differential CRISPR scores.

Supplementary Table 2

ATAC-seq data.

Supplementary Table 3

RNA-seq data.

Supplementary Table 4

List of sgRNAs and primers.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 2

Unprocessed western blot.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 3

Unprocessed western blot.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 6

Unprocessed western blot.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed western blot.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 3

Unprocessed western blot.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed western blot.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 8

Unprocessed western blot.

Source Data Extended Data Fig. 9

Statistical source data.

Source Data Extended Data Fig. 9

Unprocessed western blot.

Source Data Extended Data Fig. 10

Statistical source data.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Verma, P., Zhou, Y., Cao, Z. et al. ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination-deficient cells. Nat Cell Biol 23, 160–171 (2021). https://doi.org/10.1038/s41556-020-00624-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-020-00624-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing