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Methods Mol Biol. 2018;1813:371-387. doi: 10.1007/978-1-4939-8588-3_25.

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

Author information

1
Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA.
2
Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
3
Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA. lee.kraus@utsouthwestern.edu.
4
Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, USA. lee.kraus@utsouthwestern.edu.

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.

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)

PMID:
30097881
DOI:
10.1007/978-1-4939-8588-3_25
[Indexed for MEDLINE]

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