DNAJC9 prevents CENP-A mislocalization and chromosomal instability by maintaining the fidelity of histone supply chains.

The centromeric histone H3 variant CENP-A is overexpressed in many cancers. The mislocalization of CENP-A to noncentromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. However, pathways that promote or prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen for regulators of CENP-A localization which identified DNAJC9, a J-domain protein implicated in histone H3-H4 protein folding, as a factor restricting CENP-A mislocalization. Cells lacking DNAJC9 exhibit mislocalization of CENP-A throughout the genome, and CIN phenotypes. Global interactome analysis showed that DNAJC9 depletion promotes the interaction of CENP-A with the DNA-replication-associated histone chaperone MCM2. CENP-A mislocalization upon DNAJC9 depletion was dependent on MCM2, defining MCM2 as a driver of CENP-A deposition at ectopic sites when H3-H4 supply chains are disrupted. Cells depleted for histone H3.3, also exhibit CENP-A mislocalization. In summary, we have defined novel factors that prevent mislocalization of CENP-A, and demonstrated that the integrity of H3-H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN.

A quantitative and site-specific atlas of the citrullinome reveals widespread existence of citrullination and insights into PADI4 substrates.

Despite the importance of citrullination in physiology and disease, global identification of citrullinated proteins, and the precise targeted sites, has remained challenging. Here we employed quantitative-mass-spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites within the HL60 leukemia cell line following differentiation into neutrophil-like cells. We identified 14,056 citrullination sites within 4,008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. With this resource, we provide quantitative and site-specific information on thousands of PADI4 substrates, including signature histone marks and transcriptional regulators. Additionally, using peptide microarrays, we demonstrate the potential clinical relevance of certain identified sites, through distinct reactivities of antibodies contained in synovial fluid from anti-CCP-positive and anti-CCP-negative people with rheumatoid arthritis. Collectively, we describe the human citrullinome at a systems-wide level, provide a resource for understanding citrullination at the mechanistic level and link the identified targeted sites to rheumatoid arthritis.

Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCFß-Trcp1-dependent ubiquitylation of the actin-organizing protein Dbn1.

Ubiquitin widely modifies proteins, thereby regulating most cellular functions. The complexity of ubiquitin signalling necessitates unbiased methods enabling global detection of dynamic protein ubiquitylation. Here, we describe UBIMAX (UBiquitin target Identification by Mass spectrometry in Xenopus egg extracts), which enriches ubiquitin-conjugated proteins and quantifies regulation of protein ubiquitylation under precise and adaptable conditions. We benchmark UBIMAX by investigating DNA double-strand break-responsive ubiquitylation events, identifying previously known targets and revealing the actin-organizing protein Dbn1 as a major target of DNA damage-induced ubiquitylation. We find that Dbn1 is targeted for proteasomal degradation by the SCFß-Trcp1 ubiquitin ligase, in a conserved mechanism driven by ATM-mediated phosphorylation of a previously uncharacterized ß-Trcp1 degron containing an SQ motif. We further show that this degron is sufficient to induce DNA damage-dependent protein degradation of a model substrate. Collectively, we demonstrate UBIMAX's ability to identify targets of stimulus-regulated ubiquitylation and reveal an SCFß-Trcp1-mediated ubiquitylation mechanism controlled directly by the apical DNA damage response kinases.

PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities.

PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. We show that PARP14 is a dual-function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a marked increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13, and displays specific cellular phenotypes. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response.

The SUMO-NIP45 pathway processes toxic DNA catenanes to prevent mitotic failure.

SUMOylation regulates numerous cellular processes, but what represents the essential functions of this protein modification remains unclear. To address this, we performed genome-scale CRISPR-Cas9-based screens, revealing that the BLM-TOP3A-RMI1-RMI2 (BTRR)-PICH pathway, which resolves ultrafine anaphase DNA bridges (UFBs) arising from catenated DNA structures, and the poorly characterized protein NIP45/NFATC2IP become indispensable for cell proliferation when SUMOylation is inhibited. We demonstrate that NIP45 and SUMOylation orchestrate an interphase pathway for converting DNA catenanes into double-strand breaks (DSBs) that activate the G2 DNA-damage checkpoint, thereby preventing cytokinesis failure and binucleation when BTRR-PICH-dependent UFB resolution is defective. NIP45 mediates this new TOP2-independent DNA catenane resolution process via its SUMO-like domains, promoting SUMOylation of specific factors including the SLX4 multi-nuclease complex, which contributes to catenane conversion into DSBs. Our findings establish that SUMOylation exerts its essential role in cell proliferation by enabling resolution of toxic DNA catenanes via nonepistatic NIP45- and BTRR-PICH-dependent pathways to prevent mitotic failure.

Characterising the RNA-binding protein atlas of the mammalian brain uncovers RBM5 misregulation in mouse models of Huntington's disease.

RNA-binding proteins (RBPs) are key players regulating RNA processing and are associated with disorders ranging from cancer to neurodegeneration. Here, we present a proteomics workflow for large-scale identification of RBPs and their RNA-binding regions in the mammalian brain identifying 526 RBPs. Analysing brain tissue from males of the Huntington's disease (HD) R6/2 mouse model uncovered differential RNA-binding of the alternative splicing regulator RBM5. Combining several omics workflows, we show that RBM5 binds differentially to transcripts enriched in pathways of neurodegeneration in R6/2 brain tissue. We further find these transcripts to undergo changes in splicing and demonstrate that RBM5 directly regulates these changes in human neurons derived from embryonic stem cells. Finally, we reveal that RBM5 interacts differently with several known huntingtin interactors and components of huntingtin aggregates. Collectively, we demonstrate the applicability of our method for capturing RNA interactor dynamics in the contexts of tissue and disease.

Regulation of Rad52-dependent replication fork recovery through serine ADP-ribosylation of PolD3.

Although Poly(ADP-ribose)-polymerases (PARPs) are key regulators of genome stability, how site-specific ADP-ribosylation regulates DNA repair is unclear. Here, we describe a novel role for PARP1 and PARP2 in regulating Rad52-dependent replication fork repair to maintain cell viability when homologous recombination is dysfunctional, suppress replication-associated DNA damage, and maintain genome stability. Mechanistically, Mre11 and ATM are required for induction of PARP activity in response to replication stress that in turn promotes break-induced replication (BIR) through assembly of Rad52 at stalled/damaged replication forks. Further, by mapping ADP-ribosylation sites induced upon replication stress, we identify that PolD3 is a target for PARP1/PARP2 and that its site-specific ADP-ribosylation is required for BIR activity, replication fork recovery and genome stability. Overall, these data identify a critical role for Mre11-dependent PARP activation and site-specific ADP-ribosylation in regulating BIR to maintain genome integrity during DNA synthesis.

Serine ADP-ribosylation in Drosophila provides insights into the evolution of reversible ADP-ribosylation signalling.

In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses 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 signalling 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 dParp1:dHpf1 complex. Moreover, our structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal by Drosophila Parg. Collectively, our data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR in Animalia. The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such as Drosophila, are valuable model organisms to study the physiological role of Ser-ADPr signalling.

Transient suppression of SUMOylation in embryonic stem cells generates embryo-like structures.

Recent advances in synthetic embryology have opened new avenues for understanding the complex events controlling mammalian peri-implantation development. Here, we show that mouse embryonic stem cells (ESCs) solely exposed to chemical inhibition of SUMOylation generate embryo-like structures comprising anterior neural and trunk-associated regions. HypoSUMOylation-instructed ESCs give rise to spheroids that self-organize into gastrulating structures containing cell types spatially and functionally related to embryonic and extraembryonic compartments. Alternatively, spheroids cultured in a droplet microfluidic device form elongated structures that undergo axial organization reminiscent of natural embryo morphogenesis. Single-cell transcriptomics reveals various cellular lineages, including properly positioned anterior neuronal cell types and paraxial mesoderm segmented into somite-like structures. Transient SUMOylation suppression gradually increases DNA methylation genome wide and repressive mark deposition at Nanog. Interestingly, cell-to-cell variations in SUMOylation levels occur during early embryogenesis. Our approach provides a proof of principle for potentially powerful strategies to explore early embryogenesis by targeting chromatin roadblocks of cell fate change.

DAXX adds a de novo H3.3K9me3 deposition pathway to the histone chaperone network.

A multitude of histone chaperones are required to support histones from their biosynthesis until DNA deposition. They cooperate through the formation of histone co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using exploratory interactomics, we define the interplay between human histone H3-H4 chaperones in the histone chaperone network. We identify previously uncharacterized histone-dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX provides a unique functionality to the histone chaperone network, recruiting histone methyltransferases to promote H3K9me3 catalysis on new histone H3.3-H4 prior to deposition onto DNA. Hereby, DAXX provides a molecular mechanism for de novo H3K9me3 deposition and heterochromatin assembly. Collectively, our findings provide a framework for understanding how cells orchestrate histone supply and employ targeted deposition of modified histones to underpin specialized chromatin states.

Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry.

ADP-ribosylation is a posttranslational modification (PTM) that has crucial functions in a wide range of cellular processes. Although mass spectrometry (MS) in recent years has emerged as a valuable tool for profiling ADP-ribosylation on a system level, the use of conventional MS methods to profile ADP-ribosylation sites in an unbiased way remains a challenge. Here, we describe a protocol for identification of ADP-ribosylated proteins in vivo on a proteome-wide level, and localization of the amino acid side chains modified with this PTM. The method relies on the enrichment of ADP-ribosylated peptides using the Af1521 macrodomain (Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG, EMBO J 24:1911-1920, 2005), followed by liquid chromatography-high-resolution tandem MS (LC-MS/MS) with electron transfer dissociation-based peptide fragmentation methods, resulting in accurate localization of ADP-ribosylation sites. This protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture to data processing using the MaxQuant software suite.

SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway.

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI-FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)-mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error-free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error-free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI-deficient cells are exquisitely sensitive to ICL-inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR-mediated ICL repair is defective, and breaks are instead re-ligated by polymerase θ-dependent microhomology-mediated end-joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error-free ICL repair.

Waves of sumoylation support transcription dynamics during adipocyte differentiation.

Tight control of gene expression networks required for adipose tissue formation and plasticity is essential for adaptation to energy needs and environmental cues. However, the mechanisms that orchestrate the global and dramatic transcriptional changes leading to adipocyte differentiation remain to be fully unraveled. We investigated the regulation of nascent transcription by the sumoylation pathway during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that the sumoylation pathway has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing endogenous sumoylome dynamics in differentiating adipocytes by mass spectrometry, we found that sumoylation of specific transcription factors like PPARγ/RXR and their co-factors are associated with the transcription of adipogenic genes. Finally, using RXR as a model, we found that sumoylation may regulate adipogenic transcription by supporting the chromatin occurrence of transcription factors. Our data demonstrate that the sumoylation pathway supports the rewiring of transcriptional networks required for formation of functional adipocytes. This study also provides the scientists in the field of cellular differentiation and development with an in-depth resource of the dynamics of the SUMO-chromatin landscape, SUMO-regulated transcription and endogenous sumoylation sites during adipocyte differentiation.

ADP-ribosyltransferases, an update on function and nomenclature.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

Temporal and Site-Specific ADP-Ribosylation Dynamics upon Different Genotoxic Stresses.

The DNA damage response revolves around transmission of information via post-translational modifications, including reversible protein ADP-ribosylation. Here, we applied a mass-spectrometry-based Af1521 enrichment technology for the identification and quantification of ADP-ribosylation sites as a function of various DNA damage stimuli and time. In total, we detected 1681 ADP-ribosylation sites residing on 716 proteins in U2OS cells and determined their temporal dynamics after exposure to the genotoxins H2O2 and MMS. Intriguingly, we observed a widespread but low-abundance serine ADP-ribosylation response at the earliest time point, with later time points centered on increased modification of the same sites. This suggests that early serine ADP-ribosylation events may serve as a platform for an integrated signal response. While treatment with H2O2 and MMS induced homogenous ADP-ribosylation responses, we observed temporal differences in the ADP-ribosylation site abundances. Exposure to MMS-induced alkylating stress induced the strongest ADP-ribosylome response after 30 min, prominently modifying proteins involved in RNA processing, whereas in response to H2O2-induced oxidative stress ADP-ribosylation peaked after 60 min, mainly modifying proteins involved in DNA damage pathways. Collectively, the dynamic ADP-ribosylome presented here provides a valuable insight into the temporal cellular regulation of ADP-ribosylation in response to DNA damage.

Site-specific phosphorylation of PSD-95 dynamically regulates the postsynaptic density as observed by phase separation.

Postsynaptic density protein 95 is a key scaffolding protein in the postsynaptic density of excitatory glutamatergic neurons, organizing signaling complexes primarily via its three PSD-95/Discs-large/Zona occludens domains. PSD-95 is regulated by phosphorylation, but technical challenges have limited studies of the molecular details. Here, we genetically introduced site-specific phosphorylations in single, tandem, and full-length PSD-95 and generated a total of 11 phosphorylated protein variants. We examined how these phosphorylations affected binding to known interaction partners and the impact on phase separation of PSD-95 complexes and identified two new phosphorylation sites with opposing effects. Phosphorylation of Ser78 inhibited phase separation with the glutamate receptor subunit GluN2B and the auxiliary protein stargazin, whereas phosphorylation of Ser116 induced phase separation with stargazin only. Thus, by genetically introducing phosphoserine site-specifically and exploring the impact on phase separation, we have provided new insights into the regulation of PSD-95 by phosphorylation and the dynamics of the PSD.

Applicability of Small-Molecule Inhibitors in the Study of Peptidyl Arginine Deiminase 2 (PAD2) and PAD4.

Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline, is involved in the breakage of self-tolerance in anti-CCP-positive rheumatoid arthritis. This reaction is catalyzed by peptidyl arginine deiminases (PADs), of which PAD2 and PAD4 are thought to play key pathogenic roles. Small-molecule PAD inhibitors such as the pan-PAD inhibitor BB-Cl-amidine, the PAD2-specific inhibitor AFM-30a, and the PAD4-specific inhibitor GSK199 hold therapeutic potential and are useful tools in studies of citrullination. Using an ELISA based on the citrullination of fibrinogen, we found that AFM-30a inhibited the catalytic activity of PADs derived from live PMNs or lysed PBMCs and PMNs and of PADs in cell-free synovial fluid samples from RA patients, while GSK199 had minor effects. In combination, AFM-30a and GSK199 inhibited total intracellular citrullination and citrullination of histone H3 in PBMCs, as determined by Western blotting. They were essentially nontoxic to CD4+ T cells, CD8+ T cells, B cells, NK cells, and monocytes at concentrations ranging from 1 to 20 µM, while BB-Cl-amidine was cytotoxic at concentrations above 1 µM, as assessed by flow cytometric viability staining and by measurement of lactate dehydrogenase released from dying cells. In conclusion, AFM-30a is an efficient inhibitor of PAD2 derived from PBMCs, PMNs, or synovial fluid. AFM-30a and GSK199 can be used in combination for inhibition of PAD activity associated with PBMCs but without the cytotoxic effect of BB-Cl-amidine. This suggests that AFM-30a and GSK199 may have fewer off-target effects than BB-Cl-amidine and therefore hold greater therapeutic potential.

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

Despite 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 analyze human cells depleted for key regulators of PARP1 activity, histone PARylation factor 1 (HPF1) and ADP-ribosylhydrolase 3 (ARH3). Using quantitative proteomics, we characterize 1,596 ADP-ribosylation sites, displaying up to 1000-fold regulation across the 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-motifs, suggesting that targeting is independent of HPF1 and ARH3. Notably, we do not detect an HPF1-dependent target residue switch from serine to glutamate/aspartate under the investigated conditions. Our data support the notion that serine ADP-ribosylation mainly exists as mono-ADP-ribosylation in cells, and reveal a remarkable degree of histone co-modification with serine ADP-ribosylation and other post-translational modifications.

Serine-linked PARP1 auto-modification controls PARP inhibitor response.

Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are recruited and activated by DNA damage, resulting in ADP-ribosylation at numerous sites, both within PARP1 itself and in other proteins. Several PARP1 and PARP2 inhibitors are currently employed in the clinic or undergoing trials for treatment of various cancers. These drugs act primarily by trapping PARP1 on damaged chromatin, which can lead to cell death, especially in cells with DNA repair defects. Although PARP1 trapping is thought to be caused primarily by the catalytic inhibition of PARP-dependent modification, implying that ADP-ribosylation (ADPr) can counteract trapping, it is not known which exact sites are important for this process. Following recent findings that PARP1- or PARP2-mediated modification is predominantly serine-linked, we demonstrate here that serine ADPr plays a vital role in cellular responses to PARP1/PARP2 inhibitors. Specifically, we identify three serine residues within PARP1 (499, 507, and 519) as key sites whose efficient HPF1-dependent modification counters PARP1 trapping and contributes to inhibitor tolerance. Our data implicate genes that encode serine-specific ADPr regulators, HPF1 and ARH3, as potential PARP1/PARP2 inhibitor therapy biomarkers.

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.