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

摘要

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.