Abstract
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 scales 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 chromatin immunoprecipitation, 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.
Original language | English |
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Pages (from-to) | 474-485 |
Number of pages | 12 |
Journal | Molecular Cell |
Volume | 61 |
Issue number | 3 |
DOIs | |
Publication status | Published - 4 Feb 2016 |
Externally published | Yes |
Bibliographical note
Copyright © 2016 Elsevier Inc. All rights reserved.Keywords
- 3T3-L1 Cells
- Adenosine Diphosphate Ribose/metabolism
- Adipocytes/drug effects
- Adipogenesis
- Animals
- Binding Sites
- Cell Line, Tumor
- Cell Nucleus/drug effects
- Chromatin/genetics
- Chromatin Immunoprecipitation/methods
- Computational Biology
- Gene Expression Regulation
- Growth Hormone/pharmacology
- High-Throughput Nucleotide Sequencing
- Humans
- Hydrogen Peroxide/pharmacology
- Mice
- Oxidative Stress
- PPAR gamma/genetics
- Poly (ADP-Ribose) Polymerase-1
- Poly(ADP-ribose) Polymerases/genetics
- Promoter Regions, Genetic
- Protein Binding
- Protein Structure, Tertiary
- Transfection