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Poly(ADP-Ribose)-Dependent Chromatin Remodeling in DNA Repair

  • Théo Lebeaupin
  • Rebecca Smith
  • Sébastien Huet
  • Gyula Timinszky
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1608)

Abstract

The tightly packed and dynamic structure of chromatin can undergo major reorganization in response to endogenous or exogenous stimuli, such as the regulation of transcription or the cell cycle, or following DNA damage. A fast and local chromatin decondensation is observed upon DNA damage induced by laser micro-irradiation. This decondensation is under the control of poly(ADP-ribosyl)ation (PARylation) by PARP1, one of the first proteins recruited at the DNA damage sites. This chapter provides a step-by-step guide to perform and analyze chromatin decondensation upon DNA damage induction. The protocol is based on fluorescence microscopy of live cells expressing a core histone tagged with a photoactivatable fluorophore. Laser micro-irradiation is used to simultaneously induce DNA damage and activate the fluorescence signal within the irradiated area. This photo-perturbation experiment can be easily implemented on any confocal laser-scanning microscope equipped with a photoperturbation module. The experimental framework can also be used to follow chromatin relaxation in parallel with the recruitment kinetics of a protein of interest at DNA lesions in cells co-expressing the tagged histones and a second protein of interest fused to a different fluorescent tag.

Key words

DNA damage response Chromatin remodeling Poly(ADP-ribosyl)ation PARP1 Live-cell imaging Photo-activation 

Notes

Acknowledgements

Our work was supported by the Ligue contre le Cancer du Grand-Ouest (committees 35 and 72, to S.H.), the European Union (FP7-PEOPLE-2011-CIG, ChromaTranscript project, to S.H. and T.L.) and the Worldwide Cancer Research (#14-1315 to G.T.). Our collaboration benefited from funding from the Hubert Curien partnership/German Academic Exchange Service – DAAD – (28486ZD, to S.H., 55934632; to G.T.). The authors declare no competing financial interests.

Supplementary material

371836_2_En_12_MOESM1_ESM.docx (13 kb)
Lebeaupin-2016-Pipeline. The supplemental data contains several small files to be used as plugins for ImageJ or MathLab. The supplementary folder contains the ImageJ macros and MatLab routines required for the analysis described in Chapter  12 and a sample image stack to test the analysis routine: Poly(ADP-Ribose)-Dependent Chromatin Remodeling in DNA Repair. The ImageJ macros should be stored in the ‘ImageJ/Toolset’ folder. The three MatLab programs should be accessible for MatLab thus should be saved inside the default MatLab path. Test images have also been provided and can be used with the analysis pipeline described in Subheading 3.4. (DOCX 14 kb)

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Théo Lebeaupin
    • 1
    • 2
  • Rebecca Smith
    • 1
  • Sébastien Huet
    • 2
  • Gyula Timinszky
    • 1
  1. 1.Department of Physiological Chemistry, Biomedical Center MunichLudwig-Maximilians-Universität MünchenMartinsriedGermany
  2. 2.CNRS, UMR 6290, Institut Génétique et Développement de Rennes and Université de Rennes 1, Structure fédérative de recherche Biosit35043 Rennes CedexFrance

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