Skip to Content
Merck
  • Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.

Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.

Molecular cell (2021-05-22)
Evgeniia Prokhorova, Thomas Agnew, Anne R Wondisford, Michael Tellier, Nicole Kaminski, Danique Beijer, James Holder, Josephine Groslambert, Marcin J Suskiewicz, Kang Zhu, Julia M Reber, Sarah C Krassnig, Luca Palazzo, Shona Murphy, Michael L Nielsen, Aswin Mangerich, Dragana Ahel, Jonathan Baets, Roderick J O'Sullivan, Ivan Ahel
ABSTRACT

ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-ADPRHL2 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
DAPI, for nucleic acid staining
Sigma-Aldrich
Trichostatin A, ≥98% (HPLC), from Streptomyces sp.
Supelco
Tris(2-carboxyethyl)phosphine hydrochloride solution, 0.5 M, pH 7.0(aqueous solution; pH was adjusted with ammonium hydroxide)
Sigma-Aldrich
PDD00017273, ≥98% (HPLC)
Sigma-Aldrich
Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5, clone 6C5, Chemicon®, from mouse
Sigma-Aldrich
Anti-BRCA1 (Ab-1) Mouse mAb (MS110), liquid, clone MS110, Calbiochem®
Sigma-Aldrich
Anti-PARG Antibody, clone D8B10, clone D8B10, from mouse
Sigma-Aldrich
Deoxyribonucleic acid from calf thymus, Genomic, unsheared
Sigma-Aldrich
G 418 disulfate salt solution, 50 mg/mL in H2O, 0.1 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Anti-Histone H3 Antibody, CT, pan, serum, Upstate®
Sigma-Aldrich
Anti-pan-ADP-ribose binding reagent, from Escherichia coli
Sigma-Aldrich
Anti-Cyclin B1 Antibody, clone GNS3 (8A5D12), clone GNS3 (8A5D12), Upstate®, from mouse
Sigma-Aldrich
Anti-BRCA2 (Ab-1) Mouse mAb (2B), liquid, clone 2B, Calbiochem®
Millipore
Thymidine