Identifying genomic sites of adp-ribosylation mediated by specific nuclear parp enzymes using Click-ChIP

Ryan A. Rogge, Bryan A. Gibson, W. Lee Kraus

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages371-387
Number of pages17
DOIs
StatePublished - 2018

Publication series

NameMethods in Molecular Biology
Volume1813
ISSN (Print)1064-3745

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Rogge, R. A., Gibson, B. A., & Kraus, W. L. (2018). Identifying genomic sites of adp-ribosylation mediated by specific nuclear parp enzymes using Click-ChIP. In Methods in Molecular Biology (pp. 371-387). (Methods in Molecular Biology; Vol. 1813). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8588-3_25