Pathogenic ARH3 mutations result in ADP-ribose chromatin scars during DNA strand break repair

Abstract

Neurodegeneration is a common hallmark of individuals with hereditary defects in DNA single-strand break repair; a process regulated by poly(ADP-ribose) metabolism. Recently, mutations in the ARH3 (ADPRHL2) hydrolase that removes ADP-ribose from proteins have been associated with neurodegenerative disease. Here, we show that ARH3-mutated patient cells accumulate mono(ADP-ribose) scars on core histones that are a molecular memory of recently repaired DNA single-strand breaks. We demonstrate that the ADP-ribose chromatin scars result in reduced endogenous levels of important chromatin modifications such as H3K9 acetylation, and that ARH3 patient cells exhibit measurable levels of deregulated transcription. Moreover, we show that the mono(ADP-ribose) scars are lost from the chromatin of ARH3-defective cells in the prolonged presence of PARP inhibition, and concomitantly that chromatin acetylation is restored to normal. Collectively, these data indicate that ARH3 can act as an eraser of ADP-ribose chromatin scars at sites of PARP activity during DNA single-strand break repair.

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

“…Western blotting with anti-pan-ADPr reagent was used to assess the levels of ADPr. Consistent with our previous findings ( Fontana et al., 2017 ; Hanzlikova et al., 2020 ; Palazzo et al., 2018 ), ARH3 loss resulted in increased ADPr levels in both untreated and H 2 O 2 -treated 293T cells ( Figure 2 A), with the strongest signals corresponding to histone and PARP1 ADPr. H 2 O 2 -induced ADPr returned to baseline levels 2 h after treatment in control cells but remained elevated in ARH3-KO cells.…”

“…A short 1 h pre-treatment with the PARP1/2 inhibitor olaparib completely blocked the H 2 O 2 -induced ADPr signal in control cells, showing that this signal is PARP1/2 dependent. Conversely, the elevated basal levels of histone and PARP1 ADPr in ARH3-KO cells persisted despite 1 h pre-treatment with olaparib ( Figure 2 A) but nevertheless disappear after prolonged treatment with PARPi ( Hanzlikova et al., 2020 ).

Figure 2 Suppression of PARG activity leads to the accumulation and persistence of PARylation in ARH3-deficient cells (A) Cells were pre-treated with DMSO, 10 μM olaparib, or 10 μM PARGi for 1 h followed by 2 mM H 2 O 2 treatment for the indicated time in the presence of the drugs.

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“…The loss of ARH3 hydrolase results in the accumulation of serine-ADPr not only in response to DNA damage but also in untreated conditions ( Fontana et al., 2017 ; Hanzlikova et al., 2020 ; Palazzo et al., 2018 ). To better characterize the housekeeping role of ARH3 in the reversal of endogenous ADPr, we assessed the levels and localization of ADPr in control and ARH3-KO U2OS cells at different stages of the cell cycle.…”

The regulatory landscape of the human HPF1- and ARH3-dependent ADP-ribosylome

“…The enrichment of ADPr-modified peptides without downstream PARG treatment furthermore allowed us to infer the cellular MARylation versus PARylation equilibrium, hereby uncovering that MARylation is a global and dominant phenomenon in human cells. While observations capture the state of the cell upon lysis, and considering that ADPr is a very dynamic modification (Polo and Jackson, 2011), the detected MARylation probably stems from substrates initially being modified with PAR, which is then rapidly degraded in vivo into MAR by high hydrolase activity of eraser enzymes (Bonfiglio et al, 2020; Fontana et al, 2017; Hanzlikova et al, 2020), and PARP1 itself (Rudolph et al, 2020). Still, considering this dynamic regulation of ADPr has remained unnoticed for decades, along with the importance of better understanding the endogenous ADPr equilibrium, underscores the necessity for investigating ADPr dynamics under native conditions using unbiased proteomics strategies.…”

“…HeLa cells (CCL-2, female), U-2 OS (U2OS) cells (HTB-96, female), and HEK293T cells (CRL-3216, female), were acquired via the American Type Culture Collection, and cultured at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (Invitrogen) supplemented with 10% fetal bovine serum and a penicillin/streptomycin mixture (100 U/mL; Gibco). U2OS cells with HPF1 knockout KO (Gibbs-Seymour et al, 2016), U2OS cells with ARH3 KO (Fontana et al, 2017), HEK293T cells with HPF1 KO (Gibbs-Seymour et al, 2016), and HEK293T cells with ARH3 KO (Hanzlikova et al, 2020), were described previously. Cells were routinely tested for mycoplasma.…”