TY - JOUR
T1 - Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry.
AU - Stützer, Alexandra
AU - Welp, Luisa M
AU - Raabe, Monika
AU - Sachsenberg, Timo
AU - Kappert, Christin
AU - Wulf, Alexander
AU - Lau, Andy M
AU - David, Stefan-Sebastian
AU - Chernev, Aleksandar
AU - Kramer, Katharina
AU - Politis, Argyris
AU - Kohlbacher, Oliver
AU - Fischle, Wolfgang
AU - Urlaub, Henning
N1 - 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).
PY - 2020/10/17
Y1 - 2020/10/17
N2 - 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.
AB - 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.
UR - http://hdl.handle.net/10754/665757
UR - http://www.nature.com/articles/s41467-020-19047-7
UR - http://www.scopus.com/inward/record.url?scp=85092592945&partnerID=8YFLogxK
U2 - 10.1038/s41467-020-19047-7
DO - 10.1038/s41467-020-19047-7
M3 - Article
C2 - 33067435
SN - 2041-1723
VL - 11
JO - Nature communications
JF - Nature communications
IS - 1
ER -