Poly(ADP-ribos)ylation (PARylation) is a reversible posttranslational modification found in higher eukaryotes. However, little is known about PARylation acceptor proteins. Here, we describe a sensitive proteomics approach based on high-accuracy quantitative mass spectrometry for the identification of PARylated proteins induced under different cellular stress conditions. While confirming the majority of known PARylated substrates, our screen identifies numerous additional PARylation targets. In vivo and in vitro validation of acceptor proteins confirms that our methodology targets covalent PARylation. Nuclear proteins encompassing nucleic acid binding properties are prominently PARylated upon genotoxic stress, consistent with the nuclear localization of ARTD1/PARP1 and ARTD2/PARP2. Distinct differences in proteins becoming PARylated upon various genotoxic insults are observed, exemplified by the PARylation of RNA-processing factors THRAP3 and TAF15 under oxidative stress. High-content imaging reveals that PARylation affects the nuclear relocalization of THRAP3 and TAF15, demonstrating the potential of our approach to uncover hitherto unappreciated processes being controlled by specific genotoxic-stress-induced PARylation.

ADP-ribosylation is an important post-translational protein modification (PTM) that regulates diverse biological processes. ADP-ribosyltransferase diphtheria toxin-like 10 (ARTD10, also known as PARP10) mono-ADP-ribosylates acidic side chains and is one of eighteen ADP-ribosyltransferases that catalyze mono- or poly-ADP-ribosylation of target proteins. Currently, no enzyme is known that reverses ARTD10-catalyzed mono-ADP-ribosylation. Here we report that ARTD10-modified targets are substrates for the macrodomain proteins MacroD1, MacroD2 and C6orf130 from Homo sapiens as well as for the macrodomain protein Af1521 from archaebacteria. Structural modeling and mutagenesis of MacroD1 and MacroD2 revealed a common core structure with Asp102 and His106 of MacroD2 implicated in the hydrolytic reaction. Notably, MacroD2 reversed the ARTD10-catalyzed, mono-ADP-ribose-mediated inhibition of glycogen synthase kinase 3β (GSK3β) in vitro and in cells, thus underlining the physiological and regulatory importance of mono-ADP-ribosylhydrolase activity. Our results establish macrodomain-containing proteins as mono-ADP-ribosylhydrolases and define a class of enzymes that renders mono-ADP-ribosylation a reversible modification.

Faithful propagation of specific chromatin states requires re-establishment of epigenetic marks after every cell division. How the original epigenetic signature is inherited after disruption during DNA replication is still poorly understood. Here, we show that the poly(ADP-ribose)-polymerase-1 (PARP1/ARTD1) is implicated in the maintenance of silent rDNA chromatin during cell division. We demonstrate that PARP1 associates with TIP5, a subunit of the NoRC complex, via the noncoding pRNA and binds to silent rRNA genes after their replication in mid-late S phase. PARP1 represses rRNA transcription and is implicated in the formation of silent rDNA chromatin. Silent rDNA chromatin is a specific substrate for ADP-ribosylation and the enzymatic activity of PARP1 is necessary to establish rDNA silencing. The data unravel a function of PARP1 and ADP-ribosylation that serves to allow for the inheritance of silent chromatin structures, shedding light on how epigenetic marks are transmitted during each cell cycle.