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

Evgeniia Prokhorova,Dragana Ahel,Kang Zhu,Luca Palazzo,Ivan Ahel,Aswin Mangerich,Shona Murphy,Anne R Wondisford,James Holder,Michael L Nielsen,Roderick J O’Sullivan,Nicole Kaminski,Jonathan Baets,Danique Beijer,Thomas Agnew,Michael Tellier,Sarah C Krassnig,Joséphine Groslambert ,Marcin Suskiewicz ,Julia M Reber

doi:10.1016/j.molcel.2021.04.028

Abstract

SummaryARH3/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.

Highlights

ADP-ribosylation (ADPr) is a reversible post-translational protein modification (PTM) that regulates numerous processes, including DNA damage repair and chromatin remodeling (Gupte et al, 2017; Liu et al, 2017; Palazzo et al, 2017)
Subcellular fractionation followed by western blotting using anti-pan-ADPr reagent, which binds to all forms of cellular ADPr (Gibson et al, 2017), showed strong accumulation of ADPr in ARH3-KO but not in control cells, and the modification was predominantly detected on chromatin (Figure 1A)
Immunofluorescence analysis in cells pre-extracted with detergent prior to fixation to retain chromatin-bound proteins but remove cytoplasmic and nuclear soluble components showed the enrichment of ADPr in ARH3-KO cells and, surprisingly, revealed that ADPr persisted in interphase but even on condensed chromatin throughout different stages of mitosis (Figures 1B and 1C)

Summary

Introduction

ADP-ribosylation (ADPr) is a reversible post-translational protein modification (PTM) that regulates numerous processes, including DNA damage repair and chromatin remodeling (Gupte et al, 2017; Liu et al, 2017; Palazzo et al, 2017). PARP1 rapidly binds to DNA ends at the sites of damage and modifies itself, histones, and other proteins with mono- and poly(ADPribose) (MAR and PAR, respectively) These modifications, which promote and control DNA repair, occur predominantly on serine residues (Bonfiglio et al, 2017; Buch-Larsen et al, 2020; Hendriks et al, 2019, 2021; Leidecker et al, 2016; Palazzo et al, 2018) and, as such, require an accessory factor HPF1 for efficient synthesis (Bilokapic et al, 2020; Bonfiglio et al, 2017; Gibbs-Seymour et al, 2016; Hendriks et al, 2021; Suskiewicz et al, 2020)

Methods

Results

Discussion

Conclusion