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

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

doi:10.1038/s41467-020-19047-7

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.

Highlights

Protein–DNA interactions are key to the functionality and stability of the genome
As others and we have shown, RNA-binding sites in proteins can readily be determined by UV-induced cross-linking at 254 nm in combination with mass spectrometry (MS)[8,9,10]
The exact mass of the precursor and the masses of fragment ions of the cross-linked peptide sequence with their specific mass adducts derived from the cross-linkednucleotide moiety are searched against a sequence database[8,20]

Summary

Introduction

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 DNAdependent processes. Technologies for mapping the genome-wide distribution of histone and DNA modifications, as well as chromatin-binding proteins are well-established[6,7]. As others and we have shown, RNA-binding sites in proteins can readily be determined by UV-induced cross-linking at 254 nm in combination with mass spectrometry (MS)[8,9,10]. UV-induced cross-linking combined with MS could be a valuable technique for investigating functional and structural relations in DNA–protein systems. We introduce an experimental and computational protein–DNA cross-linking workflow suitable for simple protein–DNA complexes such as (oligo)nucleosomes, chromatin-binding factors, as well as complex systems such as cell nuclei. Our workflow allows simultaneous detection of protein cross-links to DNA and RNA in chromatin context and, provides a comprehensive picture on interactions of nucleic acids and proteins

Methods

Results

Conclusion