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Identifying Genomic Sites of ADP-Ribosylation Mediated by Specific Nuclear PARP Enzymes Using Click-ChIP

  • Ryan A. Rogge
  • Bryan A. Gibson
  • W. Lee Kraus
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)

Abstract

Nuclear poly(ADP-ribose) polymerases (PARPs), including PARPs 1, 2, and 3 and the Tankyrases, belong to a family of enzymes that can bind to chromatin and covalently modify histone- and chromatin-associated proteins with ADP-ribose derived from nuclear NAD+. The genomic loci where the nuclear PARPs bind and covalently modify chromatin are a fundamental question in PARP biology. Chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) has become an essential tool for determining specific sites of binding and modification genome-wide. Few methods are available, however, for localizing PARP-specific ADP-ribosylation events across the genome. Here we describe a variation of ChIP-seq, called Click-ChIP-seq, for identifying sites of ADP-ribosylation mediated by specific PARP family members. This method uses analog-sensitive PARP (asPARP) technology, including asPARP mutants and the alkyne-containing “clickable” NAD+ analog 8-Bu(3-yne)T-NAD+. In this assay, nuclei from cells expressing an asPARP protein of interest are incubated with 8-Bu(3-yne)T-NAD+, which is incorporated into ADP-ribose modifications mediated only by that specific asPARP protein. The nuclei are then subjected to cross-linking with formaldehyde, and the protein-linked analog ADP-ribose is clicked to biotin using copper-catalyzed alkyne-azide “click” chemistry. The chromatin is fragmented, and the fragments containing analog ADP-ribose are enriched using streptavidin-mediated precipitation. Finally, the enriched DNA is analyzed by qPCR or deep-sequencing experiments to determine which genomic loci contain ADP-ribose modifications mediated by the specific PARP protein of interest. Click-ChIP-seq has proven to be a robust and reproducible method for identifying chromatin-associated, PARP-specific ADP-ribosylation events genome-wide.

Key words

ADP-ribosylation Analog sensitivity Automodification Chromatin Chromatin immunoprecipitation (ChIP) Click chemistry Cross-link Mono(ADP-ribosyl)ation (MARylation) Mutation NAD+ analog Nucleus Nucleosome Poly(ADP-ribose) polymerase (PARP) Poly(ADP-ribosyl)ation (PARylation) Posttranslational modification (PTM) 

Notes

Acknowledgements

The PARP-related research in the Kraus Lab is supported by grants from the National Institutes of Health, NIDDK (DK069710), and the Cancer Prevention and Research Institute of Texas (CPRIT) (RP160319).

References

  1. 1.
    Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21:381–395CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Desjarlais R, Tummino PJ (2016) Role of histone-modifying enzymes and their complexes in regulation of chromatin biology. Biochemistry 55:1584–1599CrossRefPubMedGoogle Scholar
  3. 3.
    Chen P, Li G (2010) Dynamics of the higher-order structure of chromatin. Protein Cell 1:967–971CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Ame JC, Spenlehauer C, De Murcia G (2004) The PARP superfamily. BioEssays 26:882–893CrossRefPubMedGoogle Scholar
  5. 5.
    Kraus WL, Hottiger MO (2013) PARP-1 and gene regulation: progress and puzzles. Mol Asp Med 34:1109–1123CrossRefGoogle Scholar
  6. 6.
    Krishnakumar R, Kraus WL (2010) The PARP side of the nucleus: molecular actions, physiological outcomes, and clinical targets. Mol Cell 39:8–24CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Gibson BA, Kraus WL (2012) New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs. Nat Rev Mol Cell Biol 13:411–424CrossRefPubMedGoogle Scholar
  8. 8.
    Ryu KW, Kim DS, Kraus WL (2015) New facets in the regulation of gene expression by ADP-ribosylation and poly(ADP-ribose) polymerases. Chem Rev 115:2453–2481CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Vyas S, Matic I, Uchima L et al (2014) Family-wide analysis of poly(ADP-ribose) polymerase activity. Nat Commun 5:4426CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Vyas S, Chesarone-Cataldo M, Todorova T et al (2013) A systematic analysis of the PARP protein family identifies new functions critical for cell physiology. Nat Commun 4:15972CrossRefGoogle Scholar
  11. 11.
    Skidmore CJ, Davies MI, Goodwin PM et al (1979) The involvement of poly(ADP-ribose) polymerase in the degradation of NAD caused by gamma-radiation and N-methyl-N-nitrosourea. Eur J Biochem 101:135–142CrossRefPubMedGoogle Scholar
  12. 12.
    Gupte R, Liu Z, Kraus WL (2017) PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes. Genes Dev 31:101–126CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ogata N, Ueda K, Kawaichi M et al (1981) Poly(ADP-ribose) synthetase, a main acceptor of poly(ADP-ribose) in isolated nuclei. J Biol Chem 256:4135–4137PubMedGoogle Scholar
  14. 14.
    Gibson BA, Zhang Y, Jiang H et al (2016) Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation. Science 353:45–50CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Messner S, Altmeyer M, Zhao H et al (2010) PARP1 ADP-ribosylates lysine residues of the core histone tails. Nucleic Acids Res 38:6350–6362CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Ogata N, Ueda K, Kagamiyama H et al (1980) ADP-ribosylation of histone H1. Identification of glutamic acid residues 2, 14, and the COOH-terminal lysine residue as modification sites. J Biol Chem 255:7616–7620PubMedGoogle Scholar
  17. 17.
    Farrar D, Rai S, Chernukhin I et al (2010) 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 30:1199–1216CrossRefPubMedGoogle Scholar
  18. 18.
    Luo X, Ryu KW, Kim DS et al (2017) PARP-1 controls the adipogenic transcriptional program by PARylating C/EBPβ and modulating its transcriptional activity. Mol Cell 65:260–271CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Carey MF, Peterson CL, Smale ST (2009) Chromatin immunoprecipitation (ChIP). Cold Spring Harb Protoc 2009(9):pdb prot5279PubMedGoogle Scholar
  20. 20.
    Furey TS (2012) ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions. Nat Rev Genet 13:840–852CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Milne TA, Zhao K, Hess JL (2009) Chromatin immunoprecipitation (ChIP) for analysis of histone modifications and chromatin-associated proteins. Methods Mol Biol 538:409–423CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Gibson BA, Kraus WL (2017) Identification of protein substrates of specific PARP enzymes using analog-sensitive PARP mutants and a ‘clickable’ NAD+ analog. Meth Mol Biol 1608:111–135CrossRefGoogle Scholar
  23. 23.
    Presolski SI, Hong VP, Finn MG (2011) Copper-catalyzed azide-alkyne click chemistry for bioconjugation. Curr Protoc Chem Biol 3:153–162PubMedPubMedCentralGoogle Scholar
  24. 24.
    Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Robinson JT, Thorvaldsdottir H, Winckler W et al (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Thorvaldsdottir H, Robinson JT, Mesirov JP (2013) Integrative genomics viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14:178–192CrossRefPubMedGoogle Scholar
  27. 27.
    Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Ramirez F, Ryan DP, Gruning B et al (2016) deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res 44:W160–W165CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ryan A. Rogge
    • 1
    • 2
  • Bryan A. Gibson
    • 1
    • 2
  • W. Lee Kraus
    • 1
    • 2
  1. 1.Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology SciencesUniversity of Texas Southwestern Medical CenterDallasUSA
  2. 2.Division of Basic Research, Department of Obstetrics and GynecologyUniversity of Texas Southwestern Medical CenterDallasUSA

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