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

Journal: Nature communications 2020;11(1):5250

PMID: 33067435

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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.

Address: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.; Institute for Bioinformatics and Medical Informatics, University of Tübingen, 72076, Tübingen, Germany.; Applied Bioinformatics, Department for Computer Science, University of Tübingen, 72076, Tübingen, Germany.; Somatosensory Signaling and Systems Biology Group, Max Planck Institute of Experimental Medicine, 37075, Göttingen, Germany.; Department of Chemistry, King's College London, London, SE1 1DB, UK.; Laboratory of Chromatin Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.; King Abdullah University of Science and Technology (KAUST), Biological and Environmental Science and Engineering Division, Laboratory of Chromatin Biochemistry, 23955, Thuwal, Saudi Arabia.; CSL Behring GmbH, 35041, Marburg, Germany.; Institute for Translational Bioinformatics, University Hospital Tübingen, 72076, Tübingen, Germany.; Biomolecular Interactions, Max Planck Institute for Developmental Biology, 72076, Tübingen, Germany.; Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany. [email protected].; Bioanalytics Group, Institute for Clinical Chemistry, University Medical Center Göttingen, 37075, Göttingen, Germany. [email protected].

DOI: 10.1038/s41467-020-19047-7

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MeSH Terms: Chromatin, DNA, Humans, Mass Spectrometry, Nucleosomes, Polycomb-Group Proteins, Protein Binding, Proteins, Ultraviolet Rays