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

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


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) 



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).


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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
    Email author
  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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