Abstract
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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Acknowledgements
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).
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G.B. developed the ADPr-ChAP method. L.B. and G.B. performed the experiments. L.B., G.B. and M.O.H. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Compatibility of ChIP and formaldehyde fixation, and examples of unsuccessful ADPr-ChAP procedures.
(A) Exemplary results for performing chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) to investigate DNA-binding properties of GAPDH, IL1b and aSAT proteins using either anti-H3 or control IgG antibodies. Fixation was done with either 1% or 4% formaldehyde. (B) Example of unsuccessful pull-down with ADPr-ChAP due to WWE mut binding PAR (step 20 of procedure; see Troubleshooting table). (C) Example of unsuccessful ChAP-qPCR after H2O2 stimulation of A549 cells with GST-WWE wt and mut domains due to lack of enrichment of aSAT after H2O2 treatment and high backround signal from the mut (step 48 of procedure; see Troubleshooting table).
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Supplementary Tables 1–2 and Figure 1. (PDF 384 kb)
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Bisceglie, L., Bartolomei, G. & Hottiger, M. ADP-ribose-specific chromatin-affinity purification for investigating genome-wide or locus-specific chromatin ADP-ribosylation. Nat Protoc 12, 1951–1961 (2017). https://doi.org/10.1038/nprot.2017.072
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DOI: https://doi.org/10.1038/nprot.2017.072
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