Assessing kinetics and recruitment of DNA repair factors using high content screens

Barbara Martinez-Pastor; Giorgia G. Silveira; Thomas L. Clarke; Dudley Chung; Yuchao Gu; Claudia Cosentino; Lance S. Davidow; Gadea Mata; Sylvana Hassanieh; Jayme Salsman; Alberto Ciccia; Narkhyun Bae; Mark T. Bedford; Diego Megias; Lee L. Rubin; Alejo Efeyan; Graham Dellaire; Raul Mostoslavsky

doi:10.1016/j.celrep.2021.110176

Assessing kinetics and recruitment of DNA repair factors using high content screens

Barbara Martinez-Pastor, Giorgia G. Silveira, Thomas L. Clarke, Dudley Chung, Yuchao Gu, Claudia Cosentino, Lance S. Davidow, Gadea Mata, Sylvana Hassanieh, Jayme Salsman, Alberto Ciccia, Narkhyun Bae, Mark T. Bedford, Diego Megias, Lee L. Rubin, Alejo Efeyan, Graham Dellaire, Raul Mostoslavsky

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

12 Citations (Scopus)

Abstract

Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library (“ChromORFeome” library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.

Original language	English
Article number	110176
Journal	Cell Reports
Volume	37
Issue number	13
DOIs	https://doi.org/10.1016/j.celrep.2021.110176
Publication status	Published - Dec 28 2021

Bibliographical note

Funding Information:
We would like to thank all the members of the Mostoslavsky lab for helpful discussions and critical reading of the manuscript. We also thank Cari Sagum for technical help, David Root and Federica Piccioni from the Broad Institute Genetics Perturbation Platform for help with developing the ChromORFeome library, and the Bioinformatics Core, Mol. Bio. Department at MGH (NIDDK P30 DK040561). MMI laser cut technology was provided by the Program in Membrane Biology Microscopy Core, which is partially supported by the Boston Area Diabetes Endocrinology Research Center ( P30DK057521 ) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease ( P30DK043351 ). High-throughput laser-induced DNA damage was performed in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant P30 CAO13696 . B.M-P. is supported by an M + Vision Advanced Fellowship Marie Curie COFUND FP7. G.G.S. is supported by an MGH Cancer Center Excellence Award, D.C. was supported by a Nova Scotia Graduate Scholarship , and T.L.C is supported by the Charles King Trust Postdoctoral Fellowship, Simeon J. Fortin Charitable Foundation, Bank of America, N.A., Trustee. A.C. is supported by NIH grant GM117064 , and M.T.B. is supported by a CPRIT PAAC grant RP180804 . R.M. is the Laurel Schwartz Endowed Chair in Oncology. This work is supported by NIEHS grant R21ES027931 to R.M., a Natural Science and Engineering Research Council of Canada ( NSERC ) Discovery Grant RGPIN-2020-04034 to G.D, and a EU H-2020 Program Grant (ERC-2014-STG-638891) to A.E.

Funding Information:
We would like to thank all the members of the Mostoslavsky lab for helpful discussions and critical reading of the manuscript. We also thank Cari Sagum for technical help, David Root and Federica Piccioni from the Broad Institute Genetics Perturbation Platform for help with developing the ChromORFeome library, and the Bioinformatics Core, Mol. Bio. Department at MGH (NIDDK P30 DK040561). MMI laser cut technology was provided by the Program in Membrane Biology Microscopy Core, which is partially supported by the Boston Area Diabetes Endocrinology Research Center (P30DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (P30DK043351). High-throughput laser-induced DNA damage was performed in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant P30 CAO13696. B.M-P. is supported by an M + Vision Advanced Fellowship Marie Curie COFUND FP7. G.G.S. is supported by an MGH Cancer Center Excellence Award, D.C. was supported by a Nova Scotia Graduate Scholarship, and T.L.C is supported by the Charles King Trust Postdoctoral Fellowship, Simeon J. Fortin Charitable Foundation, Bank of America, N.A. Trustee. A.C. is supported by NIH grant GM117064, and M.T.B. is supported by a CPRIT PAAC grant RP180804. R.M. is the Laurel Schwartz Endowed Chair in Oncology. This work is supported by NIEHS grant R21ES027931 to R.M. a Natural Science and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN-2020-04034 to G.D, and a EU H-2020 Program Grant (ERC-2014-STG-638891) to A.E. R.M. and B.M.-P. designed the study, and R.M. B.M.-P. and G.G.S. wrote the manuscript. B.M.-P. G.G.S. T.L.C. D.C. Y.G. C.C. L.S.D. S.H. J.S. and N.B. conducted experiments. R.M. G.D. M.T.B. L.L.R. A.C. and A.E. supervised experiments. D.M. and G.M. designed and performed machine learning analysis. D.C. G.G.S. J.S. and G.D. designed and performed live imaging experiments. L.D. and L.L.R. contributed to the execution and analysis of the DNA repair screen. All authors read and edited the manuscript. The authors declare no competing interests.

Publisher Copyright:
© 2021 The Authors

ASJC Scopus Subject Areas

General Biochemistry,Genetics and Molecular Biology

PubMed: MeSH publication types

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

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10.1016/j.celrep.2021.110176

Cite this

Martinez-Pastor, B., Silveira, G. G., Clarke, T. L., Chung, D., Gu, Y., Cosentino, C., Davidow, L. S., Mata, G., Hassanieh, S., Salsman, J., Ciccia, A., Bae, N., Bedford, M. T., Megias, D., Rubin, L. L., Efeyan, A., Dellaire, G., & Mostoslavsky, R. (2021). Assessing kinetics and recruitment of DNA repair factors using high content screens. Cell Reports, 37(13), Article 110176. https://doi.org/10.1016/j.celrep.2021.110176

Martinez-Pastor, B, Silveira, GG, Clarke, TL, Chung, D, Gu, Y, Cosentino, C, Davidow, LS, Mata, G, Hassanieh, S, Salsman, J, Ciccia, A, Bae, N, Bedford, MT, Megias, D, Rubin, LL, Efeyan, A, Dellaire, G & Mostoslavsky, R 2021, 'Assessing kinetics and recruitment of DNA repair factors using high content screens', Cell Reports, vol. 37, no. 13, 110176. https://doi.org/10.1016/j.celrep.2021.110176

@article{9aaf78f5075246b5bf58962bf1c946a9,

title = "Assessing kinetics and recruitment of DNA repair factors using high content screens",

abstract = "Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library (“ChromORFeome” library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.",

author = "Barbara Martinez-Pastor and Silveira, {Giorgia G.} and Clarke, {Thomas L.} and Dudley Chung and Yuchao Gu and Claudia Cosentino and Davidow, {Lance S.} and Gadea Mata and Sylvana Hassanieh and Jayme Salsman and Alberto Ciccia and Narkhyun Bae and Bedford, {Mark T.} and Diego Megias and Rubin, {Lee L.} and Alejo Efeyan and Graham Dellaire and Raul Mostoslavsky",

note = "Funding Information: We would like to thank all the members of the Mostoslavsky lab for helpful discussions and critical reading of the manuscript. We also thank Cari Sagum for technical help, David Root and Federica Piccioni from the Broad Institute Genetics Perturbation Platform for help with developing the ChromORFeome library, and the Bioinformatics Core, Mol. Bio. Department at MGH (NIDDK P30 DK040561). MMI laser cut technology was provided by the Program in Membrane Biology Microscopy Core, which is partially supported by the Boston Area Diabetes Endocrinology Research Center ( P30DK057521 ) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease ( P30DK043351 ). High-throughput laser-induced DNA damage was performed in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant P30 CAO13696 . B.M-P. is supported by an M + Vision Advanced Fellowship Marie Curie COFUND FP7. G.G.S. is supported by an MGH Cancer Center Excellence Award, D.C. was supported by a Nova Scotia Graduate Scholarship , and T.L.C is supported by the Charles King Trust Postdoctoral Fellowship, Simeon J. Fortin Charitable Foundation, Bank of America, N.A., Trustee. A.C. is supported by NIH grant GM117064 , and M.T.B. is supported by a CPRIT PAAC grant RP180804 . R.M. is the Laurel Schwartz Endowed Chair in Oncology. This work is supported by NIEHS grant R21ES027931 to R.M., a Natural Science and Engineering Research Council of Canada ( NSERC ) Discovery Grant RGPIN-2020-04034 to G.D, and a EU H-2020 Program Grant (ERC-2014-STG-638891) to A.E. Funding Information: We would like to thank all the members of the Mostoslavsky lab for helpful discussions and critical reading of the manuscript. We also thank Cari Sagum for technical help, David Root and Federica Piccioni from the Broad Institute Genetics Perturbation Platform for help with developing the ChromORFeome library, and the Bioinformatics Core, Mol. Bio. Department at MGH (NIDDK P30 DK040561). MMI laser cut technology was provided by the Program in Membrane Biology Microscopy Core, which is partially supported by the Boston Area Diabetes Endocrinology Research Center (P30DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (P30DK043351). High-throughput laser-induced DNA damage was performed in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant P30 CAO13696. B.M-P. is supported by an M + Vision Advanced Fellowship Marie Curie COFUND FP7. G.G.S. is supported by an MGH Cancer Center Excellence Award, D.C. was supported by a Nova Scotia Graduate Scholarship, and T.L.C is supported by the Charles King Trust Postdoctoral Fellowship, Simeon J. Fortin Charitable Foundation, Bank of America, N.A. Trustee. A.C. is supported by NIH grant GM117064, and M.T.B. is supported by a CPRIT PAAC grant RP180804. R.M. is the Laurel Schwartz Endowed Chair in Oncology. This work is supported by NIEHS grant R21ES027931 to R.M. a Natural Science and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN-2020-04034 to G.D, and a EU H-2020 Program Grant (ERC-2014-STG-638891) to A.E. R.M. and B.M.-P. designed the study, and R.M. B.M.-P. and G.G.S. wrote the manuscript. B.M.-P. G.G.S. T.L.C. D.C. Y.G. C.C. L.S.D. S.H. J.S. and N.B. conducted experiments. R.M. G.D. M.T.B. L.L.R. A.C. and A.E. supervised experiments. D.M. and G.M. designed and performed machine learning analysis. D.C. G.G.S. J.S. and G.D. designed and performed live imaging experiments. L.D. and L.L.R. contributed to the execution and analysis of the DNA repair screen. All authors read and edited the manuscript. The authors declare no competing interests. Publisher Copyright: {\textcopyright} 2021 The Authors",

year = "2021",

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day = "28",

doi = "10.1016/j.celrep.2021.110176",

language = "English",

volume = "37",

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T1 - Assessing kinetics and recruitment of DNA repair factors using high content screens

AU - Martinez-Pastor, Barbara

AU - Silveira, Giorgia G.

AU - Clarke, Thomas L.

AU - Chung, Dudley

AU - Gu, Yuchao

AU - Cosentino, Claudia

AU - Davidow, Lance S.

AU - Mata, Gadea

AU - Hassanieh, Sylvana

AU - Salsman, Jayme

AU - Ciccia, Alberto

AU - Bae, Narkhyun

AU - Bedford, Mark T.

AU - Megias, Diego

AU - Rubin, Lee L.

AU - Efeyan, Alejo

AU - Dellaire, Graham

AU - Mostoslavsky, Raul

N1 - Funding Information: We would like to thank all the members of the Mostoslavsky lab for helpful discussions and critical reading of the manuscript. We also thank Cari Sagum for technical help, David Root and Federica Piccioni from the Broad Institute Genetics Perturbation Platform for help with developing the ChromORFeome library, and the Bioinformatics Core, Mol. Bio. Department at MGH (NIDDK P30 DK040561). MMI laser cut technology was provided by the Program in Membrane Biology Microscopy Core, which is partially supported by the Boston Area Diabetes Endocrinology Research Center ( P30DK057521 ) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease ( P30DK043351 ). High-throughput laser-induced DNA damage was performed in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant P30 CAO13696 . B.M-P. is supported by an M + Vision Advanced Fellowship Marie Curie COFUND FP7. G.G.S. is supported by an MGH Cancer Center Excellence Award, D.C. was supported by a Nova Scotia Graduate Scholarship , and T.L.C is supported by the Charles King Trust Postdoctoral Fellowship, Simeon J. Fortin Charitable Foundation, Bank of America, N.A., Trustee. A.C. is supported by NIH grant GM117064 , and M.T.B. is supported by a CPRIT PAAC grant RP180804 . R.M. is the Laurel Schwartz Endowed Chair in Oncology. This work is supported by NIEHS grant R21ES027931 to R.M., a Natural Science and Engineering Research Council of Canada ( NSERC ) Discovery Grant RGPIN-2020-04034 to G.D, and a EU H-2020 Program Grant (ERC-2014-STG-638891) to A.E. Funding Information: We would like to thank all the members of the Mostoslavsky lab for helpful discussions and critical reading of the manuscript. We also thank Cari Sagum for technical help, David Root and Federica Piccioni from the Broad Institute Genetics Perturbation Platform for help with developing the ChromORFeome library, and the Bioinformatics Core, Mol. Bio. Department at MGH (NIDDK P30 DK040561). MMI laser cut technology was provided by the Program in Membrane Biology Microscopy Core, which is partially supported by the Boston Area Diabetes Endocrinology Research Center (P30DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (P30DK043351). High-throughput laser-induced DNA damage was performed in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant P30 CAO13696. B.M-P. is supported by an M + Vision Advanced Fellowship Marie Curie COFUND FP7. G.G.S. is supported by an MGH Cancer Center Excellence Award, D.C. was supported by a Nova Scotia Graduate Scholarship, and T.L.C is supported by the Charles King Trust Postdoctoral Fellowship, Simeon J. Fortin Charitable Foundation, Bank of America, N.A. Trustee. A.C. is supported by NIH grant GM117064, and M.T.B. is supported by a CPRIT PAAC grant RP180804. R.M. is the Laurel Schwartz Endowed Chair in Oncology. This work is supported by NIEHS grant R21ES027931 to R.M. a Natural Science and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN-2020-04034 to G.D, and a EU H-2020 Program Grant (ERC-2014-STG-638891) to A.E. R.M. and B.M.-P. designed the study, and R.M. B.M.-P. and G.G.S. wrote the manuscript. B.M.-P. G.G.S. T.L.C. D.C. Y.G. C.C. L.S.D. S.H. J.S. and N.B. conducted experiments. R.M. G.D. M.T.B. L.L.R. A.C. and A.E. supervised experiments. D.M. and G.M. designed and performed machine learning analysis. D.C. G.G.S. J.S. and G.D. designed and performed live imaging experiments. L.D. and L.L.R. contributed to the execution and analysis of the DNA repair screen. All authors read and edited the manuscript. The authors declare no competing interests. Publisher Copyright: © 2021 The Authors

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N2 - Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library (“ChromORFeome” library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.

AB - Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library (“ChromORFeome” library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.

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Assessing kinetics and recruitment of DNA repair factors using high content screens

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