BioProject

Chromatin ADP-ribosylation regulates important cellular processes. However, the exact location and magnitude of chromatin ADP-ribosylation are largely unknown. More...

Chromatin ADP-ribosylation regulates important cellular processes. However, the exact location and magnitude of chromatin ADP-ribosylation are largely unknown. A robust and versatile method for assessing chromatin ADP-ribosylation is therefore crucial for further understanding its function. Here, we present a chromatin affinity precipitation method based on the high specificity and avidity of two well-characterized ADP-ribose binding domains to map chromatin ADP-ribosylation at the genome-wide scale and at specific loci. Our ADPr-ChAP method revealed that in cells exposed to oxidative stress, ADP-ribosylation of chromatin scaled with histone density, with highest levels at heterochromatic sites and depletion at active promoters. Furthermore, in growth factor-induced adipocyte differentiation, increased chromatin ADP-ribosylation was observed at PPARγ target genes, whose expression is ADP-ribosylation-dependent. In combination with deep-sequencing and conventional ChIP, the established ADPr-ChAP provides a valuable resource for the bioinformatic comparison of ADP-ribosylation with other chromatin modifications and for addressing its role in other biologically important processes. Overall design: Chromatin of untreated and H2O2-treated samples was pulled down with wild-type and R163A-mutant WWE domain. We performed ChAP followed by DNA-sequencinq of sheared chromatin from untreated cells enriched with wild-type RNF146 WWE domain to define the location of ADP-ribosylation in untreated cells, using the binding mutant as negative control. In addition, as oxidative stress is known to induce chromatin ADP-ribosilation, we include ChAP followed by DNA-sequencinq of sheared chromatin from H2O2-treated cells enriched with wild-type RNF146 WWE or the binding mutant. Less...