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The SUMO-NIP45 pathway guards against accumulation of toxic DNA catenanes

ABSTRACT: SUMOylation is an essential protein modification that regulates numerous biological processes, but what constitutes its most critical functions in the cell remains unclear. Here, using genome-scale CRISPR-Cas9-based synthetic lethality screens, we show that the BLM-TOP3A-RMI1-RMI2 (BTRR)-PICH pathway, which resolves ultra-fine anaphase DNA bridges (UFBs) arising from catenated DNA structures, and NIP45 (NFATC2IP) display a synthetic lethal interaction in human cells and are essential for proliferation when SUMOylation is impaired. NIP45 and SUMOylation prevent excessive UFB formation that leads to extensive binucleation when BTRR-PICH-dependent UFB resolution is defective, by orchestrating an interphase pathway for converting DNA catenanes into DNA double-strand breaks that trigger G2 arrest via canonical ATM/ATR-dependent DNA damage signaling. NIP45 exerts its crucial function in this pathway by acting as a cofactor for specific SUMOylation processes that are at least in part targeted to the SLX4 multi-nuclease complex, which may facilitate nucleolytic DNA catenane resolution. Our findings establish an essential role of SUMO signaling in underpinning cell proliferation by counteracting the deleterious threat to faithful chromosome segregation posed by toxic DNA catenanes arising in virtually every cell cycle, via non-epistatic NIP45- and BTRR-PICH-dependent pathways.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Ivo Hendriks

LAB HEAD: Michael Lund Nielsen

PROVIDER: PXD033739 | Pride | 2023-07-21

REPOSITORIES: Pride

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A SUMO-mediated protective response to DNA-protein crosslinks

Project description:DNA-protein crosslinks (DPCs) are highly cytotoxic lesions that obstruct essential DNA transactions and whose resolution is critical for cell and organismal fitness. However, unlike the repair pathways for most types of DNA damage, the mechanisms by which cells respond to and overcome DPCs remain poorly understood. Recent studies unveiled a dedicated DPC repair pathway in higher eukaryotes involving the SprT-type metalloprotease SPRTN/DVC1, which proteolytically processes DPCs during DNA replication in a ubiquitin-controlled manner. Here, we show that aldehyde-generated DPCs elicit a potent and highly dynamic, cell cycle-independent SUMOylation response impacting numerous chromatin-associated human proteins. We identify an uncharacterized SprT metalloprotease, ACRC, which unlike SPRTN is targeted to DPCs in a SUMO-dependent manner. Moreover, we establish important physiological roles of the C. elegans ACRC orthologue GCNA-1 and SUMO-mediated signaling in promoting organismal survival upon DPC formation. Finally, we demonstrate that like aldehydes, defined enzymatic DNMT1 DPCs trigger a robust SUMO response targeting the crosslinked proteins and associated factors to promote efficient lesion processing and cellular fitness. Collectively, our findings reveal an evolutionarily conserved general role of SUMO-mediated signaling in facilitating responses to DPCs in metazoans.

2019-03-26 | PXD009040 | Pride

SUMO pulldown proteomics Namalwa 5-Aza-2' deoxycytidine treatment

Project description:Combination therapies targeting malignancies aim to increase treatment efficacy and reduce toxicity. Hypomethylating drug 5-Aza-2’-deoxycytidine (5-Aza-2’) enhances transcription of tumor suppressor genes and induces replication errors via entrapment of DNMT1. Post-translational modification by SUMO plays major roles in the DNA damage response and is required for degradation of entrapped DNMT1. Here, we combine SUMOylation inhibitor TAK981 and DNA-hypomethylating agent 5-Aza-2’ to improve treatment of MYC driven hematopoietic malignancies, since MYC overexpressing tumors are sensitive to SUMOylation inhibition. We studied the classical MYC driven malignancy Burkitt lymphoma, as well as diffuse large B-cell lymphoma (DLBCL) with and without MYC translocation. SUMO inhibition prolonged the entrapment of DNMT1 to DNA, resulting in DNA damage. An increase in DNA damage was observed in cells co-treated with TAK981 and 5-Aza-2’. Both drugs synergized to reduce cell proliferation in vitro in a B cell lymphoma cell panel, including Burkitt lymphoma and DLBCL. In vivo experiments combining TAK981 (25 mg/kg) and 5-Aza-2’ (2.5 mg/kg) showed a significant reduction in outgrowth of Burkitt lymphoma in an orthotopic xenograft model. In contrast, single dosing of TAK981 was ineffective and single dosing of 5-Aza-2’ only led to a modest outgrowth reduction. TAK981 and 5-Aza-2’ synergize to reduce B cell Lymphoma outgrowth in vitro and in vivo. SUMOylation is a key-player in the repair of DNA damage, hence upon TAK981 treatment the repair of DNA damage induced by 5-Aza-2’ treatment is impaired. Our results demonstrate the potential of tailored combination of drugs, based on insight in molecular mechanisms, to improve the efficacy of cancer therapies.

2023-03-17 | PXD038620 | Pride

SUMO pulldown, U2OS, proteomics, 4 hour 20 hour 5-Aza-2' deoxycytidine treatment

2023-03-17 | PXD038617 | Pride

Targeting Pancreatic Cancer by TAK-981, a SUMOylation Inhibitor that Activates the Immune System and Blocks Tumor Cell Cycle Progression

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2022-01-10 | PXD024521 | Pride

Posttranslational modifications regulate cellular responses upon UV-induced DNA damage

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2019-10-11 | PXD010609 | Pride

SUMOYLATION AND PROTEOTOSASIS NETWORKS CONVERGE DURING HEAT SHOCK TO MINIMIZE AGGREGATION AND PROMOTE DEGRADATION OF NUCLEAR PROTEINS

Project description:The role of stress-induced increases in SUMO2/3 conjugation during the Heat Shock Response (HSR) has remained enigmatic. We investigated SUMO signal transduction at the proteomic and functional level during the HSR in the context of cells depleted of proteostasis network components via chronic Heat Shock Factor 1 inhibition versus cells with normal pro-teostasis networks. In the recovery phase post-heat shock, SUMO2/3 conjugation remained high for a prolonged time in cells lacking sufficient chaperones. Similar results were obtained upon inhibiting HSP90, indicating that increased chaperone activity during the HSR is critical for the recovery of SUMO2/3 levels post-heat shock. Proteasome inhibition during the re-covery phase likewise prolonged SUMO2/3 conjugation, indicating that stress-induced SU-MO2/3 targets are subsequently degraded by the ubiquitin-proteasome system. Functionally, we show that SUMOylation profoundly enhances solubility of target proteins upon heat shock in vitro. Collectively, our results implicate SUMO2/3 as a rapid response factor protecting the proteome from aggregation upon proteotoxic stress.

2019-03-25 | PXD010231 | Pride

Unbiased in vivo exploration of nuclear bodies-enhanced sumoylation reveals that PML orchestrates embryonic stem cell fate

Project description:Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation, but their role in pluripotency remains elusive. Here we establish that PML is required for basal SUMO2/3 conjugation in mESCs and oxidative stress-driven sumoylation in mESCs or in vivo. PML NBs create an oxidationprotective environment for UBC9-driven SUMO2/3 conjugation of PML partners, often followed by their poly-ubiquitination and degradation. Differential in vivo proteomics identified several members of the KAP1 complex as PML NB-dependent SUMO2-targets. The latter drives functional activation of this key epigenetic repressor. Accordingly, Pml-/- mESCs reexpress transposable elements and display features of totipotent-like cells, a process further enforced by PML-controlled SUMO2-conjugation of DPPA2. Finally, PML is required for adaptive stress responses in mESCs. Collectively, PML orchestrates mESC fate through SUMO2-conjugation of key transcriptional or epigenetic regulators, raising new mechanistic hypotheses about PML roles in normal or cancer stem cells.

2022-09-14 | PXD028865 | Pride

Concerted SUMO-targeted ubiquitin ligase activities of TOPORS and RNF4 are essential for stress management and cell proliferation

Project description:Protein SUMOylation provides a principal driving force for cellular stress responses including DNA-protein crosslink (DPC) repair and arsenic-induced PML body degradation. In this study, using genome-scale screens, we identify the human E3 ligase TOPORS as a key effector of SUMO-dependent DPC resolution. We demonstrate that TOPORS promotes DPC repair by functioning as a SUMO-targeted ubiquitin ligase (STUbL), combining ubiquitin ligase activity through its RING domain with poly-SUMO binding via SUMO-interacting motifs, analogous to the STUbL RNF4. Mechanistically, TOPORS is a SUMO1-selective STUbL that complements RNF4 in generating complex ubiquitin landscapes on SUMOylated targets including DPCs and PML, stimulating efficient p97/VCP unfoldase recruitment and proteasomal degradation. Combined loss of TOPORS and RNF4 is synthetic lethal even in unstressed cells, involving defective clearance of SUMOylated proteins from chromatin accompanied by cell cycle arrest and apoptosis. Our findings establish TOPORS as a STUbL whose parallel action with RNF4 defines a general mechanistic principle in crucial cellular processes governed by direct SUMO-ubiquitin crosstalk.

2024-04-11 | PXD041717 | Pride

Quantitative analysis of SUMO1 and SUMO2 proteomes in human lung A549 cells during influenza A virus infection or heat-shock

Project description:Dynamic nuclear SUMO modifications play essential roles in orchestrating cellular responses to proteotoxic stress, DNA damageand DNA virus infections. Here, we describe the host SUMOylation response to the nuclear-replicating RNA pathogen, influenz A virus. Using quantitative proteomics to compare SUMOylation responses to various stresses (including heat-shock), we reveal that influenza A virus infection causes unique re-targeting of SUMO1 and SUMO2 to a diverse range of host proteins involved in transcription, mRNA processing, RNA quality control and DNA damage repair. This global characterization of influenza virus-triggered SUMO remodeling provides a proteomic resource to understand host nuclear SUMOylation responses to infection.

2015-11-05 | PXD002943 | Pride

PML Nuclear Bodies are stress-responsive sumoylation/degradation compartments regulating KAP1 silencing function in embryonic stem cells

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2022-09-14 | PXD019609 | Pride

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