Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling. © 2023 The Author(s

The interplay of TARG1 and PARG protects against genomic instability

The timely removal of ADP-ribosylation is crucial for efficient DNA repair. However, much remains to be discovered about ADP-ribosylhydrolases. Here, we characterize the physiological role of TARG1, an ADP-ri-bosylhydrolase that removes aspartate/glutamate-linked ADP-ribosylation. We reveal its function in the DNA damage response and show that the loss of TARG1 sensitizes cells to inhibitors of topoisomerase II, ATR, and PARP. Furthermore, we find a PARP1-mediated synthetic lethal interaction between TARG1 and PARG, driven by the toxic accumulation of ADP-ribosylation, that induces replication stress and genomic instability. Finally, we show that histone PARylation factor 1 (HPF1) deficiency exacerbates the toxicity and genomic instability induced by excessive ADP-ribosylation, suggesting a close crosstalk between components of the serine-and aspartate/glutamate-linked ADP-ribosylation pathways. Altogether, our data identify TARG1 as a potential biomarker for the response of cancer cells to PARP and PARG inhibition and establish that the interplay of TARG1 and PARG protects cells against genomic instability

The interplay of TARG1 and PARG protects against genomic instability

Summary: The timely removal of ADP-ribosylation is crucial for efficient DNA repair. However, much remains to be discovered about ADP-ribosylhydrolases. Here, we characterize the physiological role of TARG1, an ADP-ribosylhydrolase that removes aspartate/glutamate-linked ADP-ribosylation. We reveal its function in the DNA damage response and show that the loss of TARG1 sensitizes cells to inhibitors of topoisomerase II, ATR, and PARP. Furthermore, we find a PARP1-mediated synthetic lethal interaction between TARG1 and PARG, driven by the toxic accumulation of ADP-ribosylation, that induces replication stress and genomic instability. Finally, we show that histone PARylation factor 1 (HPF1) deficiency exacerbates the toxicity and genomic instability induced by excessive ADP-ribosylation, suggesting a close crosstalk between components of the serine- and aspartate/glutamate-linked ADP-ribosylation pathways. Altogether, our data identify TARG1 as a potential biomarker for the response of cancer cells to PARP and PARG inhibition and establish that the interplay of TARG1 and PARG protects cells against genomic instability

TERRA-LSD1 phase separation promotes R-loop formation for telomere maintenance in ALT cancer cells

Abstract The telomere repeat-containing RNA (TERRA) forms R-loops to promote homology-directed DNA synthesis in the alternative lengthening of telomere (ALT) pathway. Here we report that TERRA contributes to ALT via interacting with the lysine-specific demethylase 1A (LSD1 or KDM1A). We show that LSD1 localizes to ALT telomeres in a TERRA dependent manner and LSD1 function in ALT is largely independent of its demethylase activity. Instead, LSD1 promotes TERRA recruitment to ALT telomeres via RNA binding. In addition, LSD1 and TERRA undergo phase separation, driven by interactions between the RNA binding properties of LSD1 and the G-quadruplex structure of TERRA. Importantly, the formation of TERRA-LSD1 condensates enriches the R-loop stimulating protein Rad51AP1 and increases TERRA-containing R-loops at telomeres. Our findings suggest that LSD1-TERRA phase separation enhances the function of R-loop regulatory molecules for ALT telomere maintenance, providing a mechanism for how the biophysical properties of histone modification enzyme-RNA interactions impact chromatin function

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

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