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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

25 Citations (Scopus)

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
Article number5250
JournalNature Communications
Volume11
Issue number1
DOIs
Publication statusPublished - 1 Dec 2020

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