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ADP-ribose-specific chromatin-affinity purification for investigating genome-wide or locus-specific chromatin ADP-ribosylation

Nature Protocols volume 12, pages 19511961 (2017) | Download Citation

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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References

  1. 1.

    & The diverse biological roles of mammalian PARPS, a small but powerful family of poly-ADP-ribose polymerases. Front. Biosci. 13, 3046–3082 (2008).

  2. 2.

    Nuclear ADP-ribosylation and its role in chromatin plasticity, cell differentiation, and epigenetics. Annu. Rev. Biochem. 84, 227–263 (2015).

  3. 3.

    & Histone ADP-ribosylation in DNA repair, replication and transcription. Trends Cell Biol. 21, 534–542 (2011).

  4. 4.

    & New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs. Nat. Rev. Mol. Cell Biol. 13, 411–424 (2012).

  5. 5.

    & PARP-1 and gene regulation: progress and puzzles. Mol. Aspects Med. 34, 1109–1123 (2013).

  6. 6.

    50 Years of poly(ADP-ribosyl)ation. Mol. Aspects Med. 34, 1043–1045 (2013).

  7. 7.

    & Activator-induced spread of poly(ADP-Ribose) polymerase promotes nucleosome loss at Hsp70. Mol. Cell 45, 64–74 (2012).

  8. 8.

    et al. Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation. Science 353, 45–50 (2016).

  9. 9.

    , , , & Analysis of chromatin ADP-ribosylation at the genome-wide level and at specific loci by ADPr-ChAP. Mol. Cell 61, 474–485 (2016).

  10. 10.

    , , , & Chromatin composition is changed by poly(ADP-ribosyl)ation during chromatin immunoprecipitation. PLoS One 7, e32914 (2012).

  11. 11.

    , & Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function. Nat. Cell Biol. 7, 1133–1139 (2005).

  12. 12.

    et al. Mutational analysis of the poly(ADP-ribosyl)ation sites of the transcription factor CTCF provides an insight into the mechanism of its regulation by poly(ADP-ribosyl)ation. Mol. Cell. Biol. 30, 1199–1216 (2010).

  13. 13.

    et al. Proteome-wide identification of the endogenous ADP-ribosylome of mammalian cells and tissue. Nat. Commun. 7, 1–13 (2016).

  14. 14.

    & An update on PARP inhibitors for the treatment of cancer. OncoTargets Ther. 8, 519–528 (2015).

  15. 15.

    et al. Chromatin proteins captured by ChIP-mass spectrometry are linked to dosage compensation in Drosophila. Nat. Struct. Mol. Biol. 20, 202–209 (2013).

  16. 16.

    et al. Identification of PARP-specific ADP-ribosylation targets reveals a regulatory function for ADP-ribosylation in transcription elongation. Mol. Cell 63, 181–183 (2016).

  17. 17.

    et al. Recognition of the iso-ADP-ribose moiety in poly(ADP-ribose) by WWE domains suggests a general mechanism for poly (ADP-ribosyl)ation-dependent ubiquitination. Genes Dev. 26, 235–240 (2012).

  18. 18.

    et al. ARTD1-induced poly-ADP-ribose formation enhances PPARγ ligand binding and co-factor exchange. Nucleic Acids Res. 43, 129–142 (2015).

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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).

Author information

Author notes

    • Giody Bartolomei

    Present address: Malcisbo AG, Schlieren, Switzerland.

    • Lavinia Bisceglie
    •  & Giody Bartolomei

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland.

    • Lavinia Bisceglie
    • , Giody Bartolomei
    •  & Michael O Hottiger
  2. Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland.

    • Lavinia Bisceglie

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael O Hottiger.

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    Supplementary Text and Figures

    Supplementary Tables 1–2 and Figure 1.

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DOI

https://doi.org/10.1038/nprot.2017.072

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