Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry.

Alexandra Stützer, Luisa M Welp, Monika Raabe, Timo Sachsenberg, Christin Kappert, Alexander Wulf, Andy M Lau, Stefan-Sebastian David, Aleksandar Chernev, Katharina Kramer, Argyris Politis, Oliver Kohlbacher, Wolfgang Fischle, Henning Urlaub

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

Protein-DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein-DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein-RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future.
Original languageEnglish (US)
JournalNature communications
Volume11
Issue number1
DOIs
StatePublished - Oct 17 2020

Bibliographical note

KAUST Repository Item: Exported on 2020-11-03
Acknowledged KAUST grant number(s): OSR-2015-CRG4-2616, OSR-2016-CRG5-3023
Acknowledgements: We thank Uwe Plessmann, Ralf Pflanz and Sabine König for excellent technical assistance in LC-MS analysis and Hossein Kohansal (Holger Stark, Dept. Structural Dynamics Max Planck Institute for Biophysical Chemistry) for HeLa cell and nuclei preparations. H.U. is supported by grants of the Deutsche Forschungsgemeinschaft (SFB860, SPP1935) and the Office of Sponsored Research of King Abdullah University of Science and Technology (OSR-2015-CRG4-2616 and OSR-2016-CRG5-3023). A.P. is funded by the Wellcome Trust (109854/Z/15/Z) and a King’s Health Partners R&D Challenge Fund through the MRC. W.F. is supported by intramural funds of the Max Planck Society and the Office of Sponsored Research of King Abdullah University of Science and Technology (OSR-2015-CRG4-2616 and OSR-2016-CRG5-3023). T.S. and O.K. acknowledge funding from the German Ministry of Research and Education (BMBF, project 031A535A, de.NBI/CIBI).

Fingerprint

Dive into the research topics of 'Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry.'. Together they form a unique fingerprint.

Cite this