Identification of mono-ADPr readers by multilevel chromatin proteomics
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: 
Thomas Colby
LAB HEAD: Ivan Matic
PROVIDER: PXD037026 | Pride | 2023-04-28
REPOSITORIES: Pride
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Project description:In the mammalian DNA damage response, ADP-ribosylation signaling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognizes damaged DNA and catalyzes the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signaling in non-mammalian Animalia. The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the Parp1:Hpf1 complex. Moreover, our structural and biochemical data reveal a new mechanism of mono-Ser-ADPr removal by Drosophila Parg.
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Project description:ADP-ribosylation (ADPr) is a reversible posttranslational modification involved in a range of cellular processes. Here, we report system-wide identification of serine ADPr in human cells upon oxidative stress. High-resolution mass spectrometry and unrestricted data processing confirm that serine residues are the major target of ADPr in HeLa cells. Proteome-wide analysis identifies 3,090 serine ADPr sites, with 97% of acceptor sites modulating more than 2-fold upon oxidative stress, while treatment with the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib abrogates this induction. Serine ADPr predominantly targets nuclear proteins, while structural-predictive analyses reveal that serine ADPr preferentially targets disordered protein regions. The identified ADP-ribosylated serines significantly overlap with known phosphorylated serines, and large-scale phosphoproteomics analysis provides evidence for the site-specific crosstalk between serine ADPr and phosphorylation. Collectively, we demonstrate that serine ADPr is a widespread modification and a major nuclear signaling response to oxidative stress, with a regulatory scope comparable to other extensive posttranslational modifications - Part 1, ADP-ribosylation data.
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Project description:Poly(ADP-ribose) polymerase 7 (PARP7) has emerged as a critically important member of a large enzyme family that catalyze mono-ADP-ribosylation (MARylation) in mammalian cells. Unlike other PARPs, PARP7 is expressed in many different cell types and is upregulated by diverse stimuli, such as toxins, growth factors, steroid hormones, and immunomodulatory ligands. Recent evidence by several independent studies show that PARP7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP7 regulates this process as well as other cellular processes. This is because the protein targets, and the specific amino acid sites, of PARP7 MARylation are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylation (ADPr) site profiling for identifying the direct targets and sites of PARP7-mediated MARylation. We found that the inactive PARP family member, PARP13—a critical regulator of the innate antiviral immune response—is a major target of PARP7, and is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide MARylation analysis reveals cysteine as a major ADPr acceptor of PARP7. These studies not only provide insight into PARP7 targeting and MARylation site preference, but also serve as an important resource for further understanding PARP7 function in the innate immune response and other pathways.
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Project description:Mono-methylation of lysine 4 on histone H3 (H3K4me1) is a well-established feature of enhancers and promoters, although its function is unknown. Remarkably, we find H3K4me1 at the promoter regions of conditionaly-repressed muscle and stimulus-dependent inflammatory response genes in myoblasts. During myogenesis, muscle genes are activated and become H3K4-trimethylated. On the promoters of active genes, we find that H3K4me1 spatially restricts the recruitment of “readers” of H3K4me3, including ING1, which, in turn, recruits Sin3A. Our findings point to a unique role for H3K4 mono-methylation in establishing boundaries that restrict the recruitment of chromatin-modifying enzymes to defined regions within promoters.
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