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A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis

Cell Death & Differentiation volume 30, pages 2078–2091 (2023)Cite this article

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Abstract

The discrimination of protein biological functions in different phases of the cell cycle is limited by the lack of experimental approaches that do not require pre-treatment with compounds affecting the cell cycle progression. Therefore, potential cycle-specific biological functions of a protein of interest could be biased by the effects of cell treatments. The OsTIR1/auxin-inducible degron (AID) system allows “on demand” selective and reversible protein degradation upon exposure to the phytohormone auxin. In the current format, this technology does not allow to study the effect of acute protein depletion selectively in one phase of the cell cycle, as auxin similarly affects all the treated cells irrespectively of their proliferation status. Therefore, the AID system requires coupling with cell synchronization techniques, which can alter the basal biological status of the studied cell population, as with previously available approaches. Here, we introduce a new AID system to Regulate OsTIR1 Levels based on the Cell Cycle Status (ROLECCS system), which induces proteolysis of both exogenously transfected and endogenous gene-edited targets in specific phases of the cell cycle. We validated the ROLECCS technology by down regulating the protein levels of TP53, one of the most studied tumor suppressor genes, with a widely known role in cell cycle progression. By using our novel tool, we observed that TP53 degradation is associated with increased number of micronuclei, and this phenotype is specifically achieved when TP53 is lost in S/G2/M phases of the cell cycle, but not in G1. Therefore, we propose the use of the ROLECCS system as a new improved way of studying the differential roles that target proteins may have in specific phases of the cell cycle.

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Fig. 1: Schematic representation of the design for Regulated OsTIR1 Levels of Expression based on the Cell Cycle Status (ROLECCS) variants.
Fig. 2: Characterization of ROLECCS AS, G1, and G2 cellular distribution and during cell-cycle progression.
Fig. 3: Biological activity of ROLECCS proteins.
Fig. 4: ROLECCS system downregulates endogenous proteins in a cell cycle-specific fashion.
Fig. 5: Cell cycle phase-specific expression and functionality of ROLECCS v2 proteins.
Fig. 6: Micronuclei accumulation upon cell cycle phase-specific TP53 abrogation.

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Data availability

Original unprocessed data used for the preparation of the manuscript are available upon kind request. All data supporting the findings of this study are available within the paper and its Supplementary Information files (Source Data File).

References

  1. Schafer KA. The cell cycle: a review. Vet Pathol. 1998;35:461–78.

    Article  CAS  PubMed  Google Scholar 

  2. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. An overview of the cell cycle. 2002.

  3. Sclafani RA, Holzen TM. Cell cycle regulation of DNA replication. Annu Rev Genet. 2007;41:237–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Takeda DY, Dutta A. DNA replication and progression through S phase. Oncogene 2005;24:2827–43.

    Article  CAS  PubMed  Google Scholar 

  5. Lew D Cell Cycle. In: Encyclopedia of Genetics. Elsevier, 2001, pp 286–96.

  6. Vermeulen K, Van Bockstaele DR, Berneman ZN. The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif. 2003;36:131–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Güttinger S, Laurell E, Kutay U. Orchestrating nuclear envelope disassembly and reassembly during mitosis. Nat Rev Mol Cell Biol. 2009;10:178–91.

    Article  PubMed  Google Scholar 

  8. Carlton JG, Jones H, Eggert US. Membrane and organelle dynamics during cell division. Nat Rev Mol Cell Biol. 2020;21:151–66.

    Article  CAS  PubMed  Google Scholar 

  9. Smoyer CJ, Jaspersen SL. Breaking down the wall: the nuclear envelope during mitosis. Curr Opin Cell Biol. 2014;26:1–9.

    Article  CAS  PubMed  Google Scholar 

  10. Salina D, Enarson P, Rattner JB, Burke B. Nup358 integrates nuclear envelope breakdown with kinetochore assembly. J Cell Biol. 2003;162:991–1001.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Raemaekers T, Ribbeck K, Beaudouin J, Annaert W, Van Camp M, Stockmans I, et al. NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization. J Cell Biol. 2003;162:1017–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. LeBrasseur N. When nuclear proteins go mitotic. J Cell Biol. 2003;162:958–9.

    PubMed Central  Google Scholar 

  13. Chao HX, Poovey CE, Privette AA, Grant GD, Chao HY, Cook JG, et al. Orchestration of DNA damage checkpoint dynamics across the human cell cycle. Cell Syst. 2017;5:445–459.e5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J. Microtubule dynamics and motor proteins during Mitosis. 2000.

  15. Schorl C, Sedivy JM. Analysis of cell cycle phases and progression in cultured mammalian cells. Methods. 2007;41:143–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Uzbekov RE. Analysis of the cell cycle and a method employing synchronized cells for study of protein expression at various stages of the cell cycle. Biochem (Mosc). 2004;69:485–96.

    Article  CAS  Google Scholar 

  17. Bittmann J, Grigaitis R, Galanti L, Amarell S, Wilfling F, Matos J et al. An advanced cell cycle tag toolbox reveals principles underlying temporal control of structure-selective nucleases. eLife 2020; 9. https://doi.org/10.7554/ELIFE.52459.

  18. Karras GI, Jentsch S. The RAD6 DNA damage tolerance pathway operates uncoupled from the replication fork and is functional beyond S phase. Cell. 2010;141:255–67.

    Article  CAS  PubMed  Google Scholar 

  19. Hombauer H, Campbell CS, Smith CE, Desai A, Kolodner RD. Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates. Cell. 2011;147:1040–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zielke N, Edgar BA. FUCCI sensors: powerful new tools for analysis of cell proliferation. Wiley Interdiscip Rev: Dev Biol. 2015;4:469–87.

    Article  CAS  PubMed  Google Scholar 

  21. Saitou T, Imamura T. Quantitative imaging with Fucci and mathematics to uncover temporal dynamics of cell cycle progression. Dev, Growth Differ. 2016;58:6–15.

    Article  PubMed  Google Scholar 

  22. Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008;132:487–98.

    Article  CAS  PubMed  Google Scholar 

  23. Vodermaier HC APC/C and SCF: Controlling each other and the cell cycle. Curr Biol. 2004; 14. https://doi.org/10.1016/j.cub.2004.09.020.

  24. Wei W, Ayad NG, Wan Y, Zhang GJ, Kirschner MW, Kaelin WG. Degradation of the SCF component Skp2 in cell-cycle phase G1 by the anaphase-promoting complex. Nature. 2004;428:194–8.

    Article  CAS  PubMed  Google Scholar 

  25. Benmaamar R, Pagano M. Involvement of the SCF complex in the control of Cdh1 degradation in S-phase. Cell Cycle. 2005;4:1230–2.

    Article  CAS  PubMed  Google Scholar 

  26. Nishitani H, Lygerou Z, Nishimoto T, Nurse P. The Cdt1 protein is required to license DNA for replication in fission yeast. Nature. 2000;404:625–8.

    Article  CAS  PubMed  Google Scholar 

  27. Sakaue-Sawano A, Yo M, Komatsu N, Hiratsuka T, Kogure T, Hoshida T, et al. Genetically encoded tools for optical dissection of the mammalian cell cycle. Mol Cell. 2017;68:626–640.e5.

    Article  CAS  PubMed  Google Scholar 

  28. Nora EP, Goloborodko A, Valton AL, Gibcus JH, Uebersohn A, Abdennur N, et al. Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell. 2017;169:930–944.e22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bajar BT, Lam AJ, Badiee RK, Oh YH, Chu J, Zhou XX, et al. Fluorescent indicators for simultaneous reporting of all four cell cycle phases. Nat Methods. 2016;13:993–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Abe T, Sakaue-Sawano A, Kiyonari H, Shioi G, Inoue KI, Horiuchi T, et al. Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter. Dev (Camb). 2013;140:237–46.

    Article  CAS  Google Scholar 

  31. Spencer SL, Cappell SD, Tsai F-C, Overton KW, Wang CL, Meyer T. The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit. Cell. 2013;155:369–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Anzalone AV, Koblan LW, Liu DR. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol. 2020;38:824–44.

    Article  CAS  PubMed  Google Scholar 

  33. Ashfaq MA, Dinesh Kumar V, Soma Sekhar Reddy P, Anil Kumar C, Sai Kumar K, Narasimha Rao N, et al. Post-transcriptional gene silencing: basic concepts and applications. J Biosci. 2020;45:1–10.

    Article  Google Scholar 

  34. Kallunki T, Barisic M, Jäättelä M, Liu B. How to choose the right inducible gene expression system for mammalian studies? Cells. 2019; 8. https://doi.org/10.3390/cells8080796.

  35. Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano R, et al. The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice. Nat Commun. 2020;11:1–13.

    Article  Google Scholar 

  36. Röth S, Fulcher LJ, Sapkota GP. Advances in targeted degradation of endogenous proteins. Cell Mol Life Sci. 2019;76:2761–77.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT. Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Rep. 2016;15:210–8.

    Article  CAS  PubMed  Google Scholar 

  38. Nishimura H, Nose M, Hiai H, Minato N, Honjo T. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999;11:141–51.

    Article  CAS  PubMed  Google Scholar 

  39. Kubota T, Nishimura K, Kanemaki MT, Donaldson AD. The Elg1 replication factor c-like complex functions in PCNA unloading during DNA replication. Mol Cell. 2013;50:273–80.

    Article  CAS  PubMed  Google Scholar 

  40. Hoffmann S, Fachinetti D. Real-time de novo deposition of centromeric histone-associated proteins using the auxin-inducible degradation system. In: Methods Mol Biol. Humana Press Inc., 2018, pp 223-41.

  41. Lai S-L, Perng R-P, Hwang J. p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells. J Biomed Sci. 2000;7:64–70.

    Article  CAS  PubMed  Google Scholar 

  42. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8:2281–308.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Gutschner T, Haemmerle M, Genovese G, Draetta GF, Chin L. Post-translational regulation of Cas9 during G1 enhances homology-directed repair. Cell Rep. 2016;14:1555–66.

    Article  CAS  PubMed  Google Scholar 

  44. Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev. 2010;90:1103–63.

    Article  CAS  PubMed  Google Scholar 

  45. Terpe K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2003;60:523–33.

    Article  CAS  PubMed  Google Scholar 

  46. Yesbolatova A, Saito Y, Kanemaki MT. Constructing auxin-inducible degron mutants using an all-in-one vector. Pharmaceuticals 2020; 13. https://doi.org/10.3390/ph13050103.

  47. Kim JH, Lee S-R, Li L-H, Park H-J, Park J-H, Lee KY, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE. 2011;6:e18556.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Liu E, Li X, Yan F, Zhao Q, Wu X. Cyclin-dependent kinases phosphorylate human Cdt1 and induce its degradation. J Biol Chem. 2004;279:17283–8.

    Article  CAS  PubMed  Google Scholar 

  49. Vassilev LT, Tovar C, Chen S, Knezevic D, Zhao X, Sun H, et al. Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci USA. 2006;103:10660.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Vousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol. 2007;8:275–83.

    Article  CAS  PubMed  Google Scholar 

  51. Kastenhuber ER, Lowe SW. Putting p53 in Context. Cell 2017;170:1062–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Levine AJ p53: 800 million years of evolution and 40 years of discovery. https://doi.org/10.1038/s41568-020-0262-1.

  53. Huang L, Pike D, Sleat DE, Nanda V, Lobel P. Potential pitfalls and solutions for use of fluorescent fusion proteins to study the lysosome. PLoS ONE 2014; 9. https://doi.org/10.1371/journal.pone.0088893.

  54. Yesbolatova A, Natsume T, Hayashi KI, Kanemaki MT. Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole. Methods. 2019;164–165:73–80.

    Article  PubMed  Google Scholar 

  55. Li S, Prasanna X, Salo VT, Vattulainen I, Ikonen E. An efficient auxin-inducible degron system with low basal degradation in human cells. Nat Methods. 2019;16:866–9.

    Article  CAS  PubMed  Google Scholar 

  56. Mantovani F, Collavin L, Del Sal G. Mutant p53 as a guardian of the cancer cell. Cell Death Differ. 2019;26:199–212.

    Article  PubMed  Google Scholar 

  57. Lane DP. Cancer. p53, guardian of the genome. Nature. 1992;358:15–6.

    Article  CAS  PubMed  Google Scholar 

  58. Hatch EM, Fischer AH, Deerinck TJ, Hetzer MW. Catastrophic nuclear envelope collapse in cancer cell micronuclei. Cell. 2013;154:47–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Salazar AM, Sordo M, Ostrosky-Wegman P. Relationship between micronuclei formation and p53 induction. Mutat Res. 2009;672:124–8.

    Article  CAS  PubMed  Google Scholar 

  60. Thompson SL, Compton DA. Proliferation of aneuploid human cells is limited by a p53-dependent mechanism. J Cell Biol. 2010;188:369–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Kakoti S, Yamauchi M, Gu W, Kato R, Yasuhara T, Hagiwara Y, et al. p53 deficiency augments nucleolar instability after ionizing irradiation. Oncol Rep. 2019;42:2293–302.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Kadonaga JT. Regulation of RNA polymerase II transcription by sequence-specific DNA binding factors. Cell. 2004;116:247–57.

    Article  CAS  PubMed  Google Scholar 

  63. Lambrus BG, Moyer TC, Holland AJ. Applying the auxin-inducible degradation system for rapid protein depletion in mammalian cells. In: Methods Cell Biol. Academic Press Inc., 2018, pp 107–35.

  64. Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods. 2009;6:917–22.

    Article  CAS  PubMed  Google Scholar 

  65. Grant GD, Kedziora KM, Limas JC, Cook JG, Purvis JE. Accurate delineation of cell cycle phase transitions in living cells with PIP-FUCCI. Cell Cycle (Georget, Tex). 2018;17:2496–516.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Dr. Emanuele Cocucci for the scientific discussions during the preparation of this manuscript. We also thank the countless investigators who made their plasmids and reagents available through public repositories such as Addgene.

Funding

This work was supported by seed funds from The Ohio State University Comprehensive Cancer Center (DP, CMC), Pelotonia (DP), NCI-NIH (NIH R35CA197706) (CMC), and ORIP-NIH (K01OD031811-01) (AED). The Gene Editing Shared Resource, the Flow Cytometry Shared Resource and the Genomics Shared Resource that contributed to this study are supported by the Cancer Center Support Grant P30CA016058.

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Author notes

  1. These authors contributed equally: Dario Palmieri, Carlo M. Croce.

Authors and Affiliations

  1. Department of Cancer Biology and Genetics, College of Medicine, The Ohio State University, 43210, Columbus, OH, USA

    Marina Capece, Anna Tessari, Joseph Mills, Gian Luca Rampioni Vinciguerra, Chenyu Lin, Bryan K. McElwain, Wayne O. Miles, Vincenzo Coppola, Dario Palmieri & Carlo M. Croce

  2. The Ohio State University Wexner Medical Center and Comprehensive Cancer Center, 43210, Columbus, OH, USA

    Marina Capece, Anna Tessari, Joseph Mills, Gian Luca Rampioni Vinciguerra, Darian Louke, Chenyu Lin, Bryan K. McElwain, Wayne O. Miles, Vincenzo Coppola, Alexander E. Davies, Dario Palmieri & Carlo M. Croce

  3. Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 43210, Columbus, OH, USA

    Darian Louke & Alexander E. Davies

  4. Gene Editing Shared Resource, The Ohio State University Wexner Medical Center and Comprehensive Cancer Center, 43210, Columbus, OH, USA

    Dario Palmieri

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Contributions

MC, DP and CMC conceived and designed the project. MC, DP, AT, WOM conceived and planned the experiments. MC, DP, AT, JM, GLRV, CL, WOM executed the experiments. AED and DL performed and analyzed live microscopy experiments. MC, DP, AT, GLRV, CL, WOM and AED analyzed the data. BM provided technical support for flow cytometry experiments and cell sorting. MC and DP, wrote the manuscript. AT, JM, GLRV, CL, WOM, VC and CMC provided critical feedback and contributed to the final version of the manuscript.

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Correspondence to Dario Palmieri or Carlo M. Croce.

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Capece, M., Tessari, A., Mills, J. et al. A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis. Cell Death Differ 30, 2078–2091 (2023). https://doi.org/10.1038/s41418-023-01191-4

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