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

Abstract

SUMMARYDespite the involvement of Poly(ADP-ribose) polymerase-1 (PARP1) in many important biological pathways, the target residues of PARP1-mediated ADP-ribosylation remain ambiguous. To explicate the ADP-ribosylation regulome, we analyzed human cells depleted for key regulators of PARP1 activity, histone PARylation factor 1 (HPF1) and ADP-ribosylhydrolase 3 (ARH3). Using quantitative proteomics, we quantified 1,596 ADPr sites, displaying a thousand-fold regulation across investigated knockout cells. We find that HPF1 and ARH3 inversely and homogenously regulate the serine ADP-ribosylome on a proteome-wide scale with consistent adherence to lysine-serine (KS)-motifs suggesting targeting is independent of HPF1 and ARH3. Our data reveal that ADPr globally exists as mono-ADP-ribosylation, and we detail a remarkable degree of histone co-modification with ADPr and other post-translational modifications. Strikingly, no HPF1-dependent target residue switch from serine to glutamate/aspartate was detectable in cells, which challenges current dogma related to PARP1 target residues. Collectively, we elucidate hitherto unappreciated processes related to cellular PARP1 activity.

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

“…Combined with previous findings ( Abplanalp et al., 2017 ; Fontana et al., 2017 ; Hanzlikova et al., 2020 ; Hendriks et al., 2021 ; Palazzo et al., 2018 ), our data confirm that ARH3 is the main hydrolase of endogenous serine-linked MAR ( Figures 6 A and 6B), while PARG swiftly removes long PAR chains ( Figures 6 A and 6C) that are composed largely of PARP1 autoPARylation in an unstressed environment but could also come from the DNA replication-associated events, namely unligated Okazaki fragments ( Hanzlikova et al., 2018 ). Critically, whereas elevated MARylation in ARH3-deficient cells is well tolerated, combined ARH3 deficiency and PARG suppression results in the accumulation and persistence of PARylation that is highly toxic to the cell and has distinct (patho)physiological effects ( Figure 6 D).…”

“…Subcellular fractionation following cell cycle synchronization confirmed these observations ( Figures S1 A and S1B). The signals were lost upon small interfering RNA (siRNA)-mediated HPF1 knockdown ( Figures S1 C–S1E), which together with a recent mass spectrometry study ( Hendriks et al., 2021 ) confirms that ADPr in ARH3-KO U2OS cells is specifically enriched at serine residues.

Figure 1 Loss of ARH3 leads to the enrichment of chromatin-associated MARylation throughout the cell cycle (A) Cells were subjected to subcellular fractionation.

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“…Consequently, using ARH3-deficient cells, we were able, for the first time, to demonstrate that serine-linked MAR and possibly short PAR is generated at all stages of the cell cycle and is largely associated with chromatin ( Figure 6 B). Indeed, many chromatin and chromatin-binding proteins were identified as serine-ADPr substrates by mass spectrometry ( Bonfiglio et al., 2017 ; Buch-Larsen et al., 2020 ; Hendriks et al., 2019 , 2021 ; Larsen et al., 2018 ). Thus, non-toxic suppression of ARH3 can be a valuable tool to study the many regulatory functions of protein ADPr under undamaged conditions.…”

“…PARP1 rapidly binds to DNA ends at the sites of damage and modifies itself, histones, and other proteins with mono- and poly(ADP-ribose) (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 ).…”

Uncovering the Invisible: Mono-ADP-ribosylation Moved into the Spotlight

“…Those and similar peptides and proteins carrying different ADP-ribose-like units indeed enabled the successful generation of several polyclonal ADP-ribose-recognizing antibodies [127,155]. A comparison between some of these antibodies and the macrodomain Af1521 commonly used for MS analysis, however, revealed that for those analysis, the macrodomain still outperforms the antibodies [174]. Nonetheless, while the new antibodies recognize MAR and PAR and therefore are referred to as pan-ADP-ribose antibodies, they could already be used to study organelle and mono-ART-specific ADP-ribosylation dynamics by Western blotting (WB) and Immunofluorescence (IF) in different contexts [127,175].…”