The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8

Abhay Narayan Singh; Judith Oehler; Ignacio Torrecilla; Susan Kilgas; Shudong Li; Bruno Vaz; Claire Guérillon; John Fielden; Esperanza Hernandez-Carralero; Elisa Cabrera; Iain D.C. Tullis; Mayura Meerang; Paul R. Barber; Raimundo Freire; Jason Parsons; Borivoj Vojnovic; Anne E. Kiltie; Niels Mailand; Kristijan Ramadan

doi:10.15252/embj.2019102361

The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8

Abhay Narayan Singh, Judith Oehler, Ignacio Torrecilla, Susan Kilgas, Shudong Li, Bruno Vaz, Claire Guérillon, John Fielden, Esperanza Hernandez-Carralero, Elisa Cabrera, Iain D.C. Tullis, Mayura Meerang, Paul R. Barber, Raimundo Freire, Jason Parsons, Borivoj Vojnovic, Anne E. Kiltie, Niels Mailand, Kristijan Ramadan^* (Corresponding Author)

^*Corresponding author for this work

The Rowett Institute of Nutrition and Health

Research output: Contribution to journal › Article › peer-review

31 Citations (Scopus)

Abstract

The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97–ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97–ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97–ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.

Original language	English
Article number	e102361
Number of pages	25
Journal	EMBO Journal
Volume	38
Issue number	21
Early online date	15 Oct 2019
DOIs	https://doi.org/10.15252/embj.2019102361
Publication status	Published - 4 Nov 2019

Bibliographical note

Funding Information:
The Ramadan Laboratory is supported by a Medical Research Council UK programme grant (MC_EX_MR/K022830/1) to K.R. J.O. was supported by a Swiss National Science Foundation grant (31003A_141197) to K.R., a Goodger and Schorstein Scholarship, University of Oxford, and a European Institute of Innovation and Technology short-term post-doctoral fellowship. S. K, J. F. and S. L were supported by the Cancer Research UK or MRC studentships. B. Vo., A. E. K. and I. D. C. T. are supported by Cancer Research UK Programme Grant C5255/A15935. C.G. was supported by the Danish Cancer Society (Grant No. R146-RP11394). R. F. is supported by Spanish Ministry of Innovation Science and Universities/EU-ERDF (SAF2016-80626-R), E. C. is supported by the Fundaci?n DISA, and E. H-C. is supported by an ACIISI pre-doctoral fellowship. We thank Ross Chapman for providing us with WT and 53BP1-deficient MCF7 cell lines. We are grateful to Pam Reynolds for support with the UV micro-irradiation system, to the Mechanical and Electronics Workshops (J Prentice and RG Newman) for assistance with construction of the system, to the Bioanalysis Core (Graham Brown and Ioland Vendrell) for the supports with microscopy and mass spectrometry and to the Radiation Biophysics Core for the help with X-ray radiation. We thank Benedikt Kessler for his constant support with mass spectrometry. We thank Jeremy Stark for providing us with GFP-reporter assays for HR and NHEJ.

Data Availability Statement

No data availability statement.

Keywords

Ataxin 3
DNA double-strand break repair
E3 ubiquitin ligase RNF8
genome stability
p97/VCP ATPase

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Singh, A. N., Oehler, J., Torrecilla, I., Kilgas, S., Li, S., Vaz, B., Guérillon, C., Fielden, J., Hernandez-Carralero, E., Cabrera, E., Tullis, I. D. C., Meerang, M., Barber, P. R., Freire, R., Parsons, J., Vojnovic, B., Kiltie, A. E., Mailand, N., & Ramadan, K. (2019). The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8. EMBO Journal, 38(21), Article e102361. https://doi.org/10.15252/embj.2019102361

Singh, AN, Oehler, J, Torrecilla, I, Kilgas, S, Li, S, Vaz, B, Guérillon, C, Fielden, J, Hernandez-Carralero, E, Cabrera, E, Tullis, IDC, Meerang, M, Barber, PR, Freire, R, Parsons, J, Vojnovic, B, Kiltie, AE, Mailand, N & Ramadan, K 2019, 'The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8', EMBO Journal, vol. 38, no. 21, e102361. https://doi.org/10.15252/embj.2019102361

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title = "The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8",

abstract = "The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97–ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97–ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97–ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.",

keywords = "Ataxin 3, DNA double-strand break repair, E3 ubiquitin ligase RNF8, genome stability, p97/VCP ATPase",

author = "Singh, {Abhay Narayan} and Judith Oehler and Ignacio Torrecilla and Susan Kilgas and Shudong Li and Bruno Vaz and Claire Gu{\'e}rillon and John Fielden and Esperanza Hernandez-Carralero and Elisa Cabrera and Tullis, {Iain D.C.} and Mayura Meerang and Barber, {Paul R.} and Raimundo Freire and Jason Parsons and Borivoj Vojnovic and Kiltie, {Anne E.} and Niels Mailand and Kristijan Ramadan",

note = "Funding Information: The Ramadan Laboratory is supported by a Medical Research Council UK programme grant (MC_EX_MR/K022830/1) to K.R. J.O. was supported by a Swiss National Science Foundation grant (31003A_141197) to K.R., a Goodger and Schorstein Scholarship, University of Oxford, and a European Institute of Innovation and Technology short-term post-doctoral fellowship. S. K, J. F. and S. L were supported by the Cancer Research UK or MRC studentships. B. Vo., A. E. K. and I. D. C. T. are supported by Cancer Research UK Programme Grant C5255/A15935. C.G. was supported by the Danish Cancer Society (Grant No. R146-RP11394). R. F. is supported by Spanish Ministry of Innovation Science and Universities/EU-ERDF (SAF2016-80626-R), E. C. is supported by the Fundaci?n DISA, and E. H-C. is supported by an ACIISI pre-doctoral fellowship. We thank Ross Chapman for providing us with WT and 53BP1-deficient MCF7 cell lines. We are grateful to Pam Reynolds for support with the UV micro-irradiation system, to the Mechanical and Electronics Workshops (J Prentice and RG Newman) for assistance with construction of the system, to the Bioanalysis Core (Graham Brown and Ioland Vendrell) for the supports with microscopy and mass spectrometry and to the Radiation Biophysics Core for the help with X-ray radiation. We thank Benedikt Kessler for his constant support with mass spectrometry. We thank Jeremy Stark for providing us with GFP-reporter assays for HR and NHEJ.",

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doi = "10.15252/embj.2019102361",

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T1 - The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8

AU - Singh, Abhay Narayan

AU - Oehler, Judith

AU - Torrecilla, Ignacio

AU - Kilgas, Susan

AU - Li, Shudong

AU - Vaz, Bruno

AU - Guérillon, Claire

AU - Fielden, John

AU - Hernandez-Carralero, Esperanza

AU - Cabrera, Elisa

AU - Tullis, Iain D.C.

AU - Meerang, Mayura

AU - Barber, Paul R.

AU - Freire, Raimundo

AU - Parsons, Jason

AU - Vojnovic, Borivoj

AU - Kiltie, Anne E.

AU - Mailand, Niels

AU - Ramadan, Kristijan

N1 - Funding Information: The Ramadan Laboratory is supported by a Medical Research Council UK programme grant (MC_EX_MR/K022830/1) to K.R. J.O. was supported by a Swiss National Science Foundation grant (31003A_141197) to K.R., a Goodger and Schorstein Scholarship, University of Oxford, and a European Institute of Innovation and Technology short-term post-doctoral fellowship. S. K, J. F. and S. L were supported by the Cancer Research UK or MRC studentships. B. Vo., A. E. K. and I. D. C. T. are supported by Cancer Research UK Programme Grant C5255/A15935. C.G. was supported by the Danish Cancer Society (Grant No. R146-RP11394). R. F. is supported by Spanish Ministry of Innovation Science and Universities/EU-ERDF (SAF2016-80626-R), E. C. is supported by the Fundaci?n DISA, and E. H-C. is supported by an ACIISI pre-doctoral fellowship. We thank Ross Chapman for providing us with WT and 53BP1-deficient MCF7 cell lines. We are grateful to Pam Reynolds for support with the UV micro-irradiation system, to the Mechanical and Electronics Workshops (J Prentice and RG Newman) for assistance with construction of the system, to the Bioanalysis Core (Graham Brown and Ioland Vendrell) for the supports with microscopy and mass spectrometry and to the Radiation Biophysics Core for the help with X-ray radiation. We thank Benedikt Kessler for his constant support with mass spectrometry. We thank Jeremy Stark for providing us with GFP-reporter assays for HR and NHEJ.

PY - 2019/11/4

Y1 - 2019/11/4

N2 - The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97–ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97–ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97–ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.

AB - The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97–ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97–ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97–ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.

KW - Ataxin 3

KW - DNA double-strand break repair

KW - E3 ubiquitin ligase RNF8

KW - genome stability

KW - p97/VCP ATPase

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DO - 10.15252/embj.2019102361

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SN - 0261-4189

VL - 38

JO - EMBO Journal

JF - EMBO Journal

IS - 21

M1 - e102361

ER -

The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8

Abstract

Bibliographical note

Data Availability Statement

Keywords

UN SDGs

Access to Document

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