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

Nat Commun. 2020 Oct 16;11(1):5250. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatin / chemistry
  • Chromatin / genetics
  • Chromatin / metabolism*
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism*
  • DNA / radiation effects*
  • Humans
  • Mass Spectrometry
  • Nucleosomes / chemistry
  • Nucleosomes / genetics
  • Nucleosomes / metabolism
  • Polycomb-Group Proteins / chemistry
  • Polycomb-Group Proteins / genetics
  • Polycomb-Group Proteins / metabolism
  • Polycomb-Group Proteins / radiation effects
  • Protein Binding / radiation effects
  • Proteins / chemistry
  • Proteins / genetics
  • Proteins / metabolism*
  • Proteins / radiation effects
  • Ultraviolet Rays

Substances

  • Chromatin
  • Nucleosomes
  • Polycomb-Group Proteins
  • Proteins
  • SCML2 protein, human
  • DNA