Poly(ADP-Ribose)-Dependent Chromatin Remodeling in DNA Repair
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 wordsDNA damage response Chromatin remodeling Poly(ADP-ribosyl)ation PARP1 Live-cell imaging Photo-activation
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.
- 3.Wei H, Yu X (2016) Functions of PARylation in DNA damage repair pathways. Genomics Proteomics & Bioinformatics 14(3):131-139.Google Scholar
- 5.Timinszky G, Till S, Hassa PO, Hothorn M, Kustatscher G, Nijmeijer B, Colombelli J, Altmeyer M, Stelzer EHK, Scheffzek K, Hottiger MO, Ladurner AG (2009) A macrodomain-containing histone rearranges chromatin upon sensing PARP1 activation. Nat Struct Mol Biol 16(9):923–U941CrossRefPubMedGoogle Scholar
- 7.Neumann B, Walter T, Heriche JK, Bulkescher J, Erfle H, Conrad C, Rogers P, Poser I, Held M, Liebel U, Cetin C, Sieckmann F, Pau G, Kabbe R, Wunsche A, Satagopam V, Schmitz MHA, Chapuis C, Gerlich DW, Schneider R, Eils R, Huber W, Peters JM, Hyman AA, Durbin R, Pepperkok R, Ellenberg J (2010) Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes. Nature 464(7289):721–727CrossRefPubMedPubMedCentralGoogle Scholar
- 12.McNally JG, Smith CL (2001) Photobleaching by confocal microscopy. In: Diaspro A (ed) Confocol and two-photon microscopy: foundations, applications and advances. Wiley, Hoboken, NJ, pp 525–538Google Scholar
- 13.Bancaud A, Huet S, Rabut G, Ellenberg J (2010) Fluorescence perturbation techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, photoactivation, photoconversion, and FLIP. In: Goldman RD, Swedlow JR, Spector DL (eds) Live cell imaging: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- 15.Thevenaz P, Unser M (1998) An efficient mutual information optimizer for multiresolution image registration. 1998 International Conference on Image Processing - Proceedings, Vol 1. Chicago, ILGoogle Scholar