Protein ADP-ribosylation is a structurally heterogeneous post-translational modification (PTM) that influences the physicochemical and biological properties of the modified protein. ADP-ribosylation of chromatin changes its structural properties, thereby regulating important nuclear functions. A lack of suitable antibodies for chromatin immunoprecipitation (ChIP) has so far prevented a comprehensive analysis of DNA-associated protein ADP-ribosylation. To analyze chromatin ADP-ribosylation, we recently developed a novel ADP-ribose-specific chromatin-affinity purification (ADPr-ChAP) methodology that uses the recently identified ADP-ribose-binding domains RNF146 WWE and Af1521. In this protocol, we describe how to use this robust and versatile method for genome-wide and loci-specific localization of chromatin ADP-ribosylation. ADPr-ChAP enables bioinformatic comparisons of ADP-ribosylation with other chromatin modifications and is useful for understanding how ADP-ribosylation regulates biologically important cellular processes. ADPr-ChAP takes 1 week and requires standard skills in molecular biology and biochemistry. Although not covered in detail here, this technique can also be combined with conventional ChIP or DNA analysis to define the histone marks specifically associated with the ADP-ribosylated chromatin fractions and dissect the molecular mechanism and functional role of chromatin ADP-ribosylation.
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We thank V. Bilan and M. Lehmann (Department of Molecular Mechanisms of Disease, University of Zurich) for helpful advice and comments regarding the development of the method; M. Leutert (Department of Molecular Mechanisms of Disease, University of Zurich) for advice on graphics; and S. Christen, T. Suter and D.L. Pedrioli (Department of Molecular Mechanisms of Disease, University of Zurich) for editorial assistance and critical input during writing. We thank the Functional Genomics Centre Zurich for access to the computational infrastructure. ADP-ribosylation research in the laboratory of M.O.H. was funded by the Canton of Zurich, the University Research Priority Program (URPP) in Translational Cancer Biology at the University of Zurich, and the Swiss National Science Foundation (grants 310030B_138667 and 310030_157019).
Integrated supplementary information
Supplementary Tables 1–2 and Figure 1.