Treacle-dependent TOPBP1 condensation regulates the nucleolar DNA damage response.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE316591 | GEO | 2026/05/27
REPOSITORIES: GEO
Similar Datasets
Project description:Ribosomal DNA (rDNA) arrays are highly repetitive regions of the genome which encode essential genes required to produce ribosomes. DNA double-stranded breaks (DSBs) generated within rDNA genes elicit a unique cellular response involving robust transcriptional silencing and nucleolar reorganization into ‘cap’ structures at the nucleolar periphery. This process is coordinated by the nucleolar scaffolding protein TCOF1, which functions to recruit the DNA repair proteins NBS1 and TOPBP1 that activate the ATM and ATR kinases, resulting in ribosomal RNA (rRNA) transcriptional silencing and nucleolar segregation. However, the DNA damage and repair response at rDNA arrays remains incompletely understood. Here, we investigate the cellular response to rDNA DSBs using proteomics and genetic CRISPR-Cas9 screening. We show that the protein UFMylation pathway and the HUSH complex are important for cell viability and survival in response to rDNA DSBs, and that the E3 UFM1-ligase UFL1 and its heterodimer DDRGK1 are associated with TCOF1 at nucleolar caps. Loss of UFL1 leads to impaired ATM activation, reduced rRNA transcriptional silencing, and an overall reduction in nucleolar segregation. We identified ATM, UNC45A and SMC6 as UFMylated proteins, in which UFMylation may facilitate ATM activation and segregation of damaged rDNA to the nucleolar periphery. Altogether, our findings provide the first evidence for a role for UFMylation in rDNA DSB repair.
2024-01-29 | GSE253809 | GEO
Project description:Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.
2019-12-06 | PXD015138 | Pride
Project description:Ribosomal DNA (rDNA) arrays are highly repetitive regions of the genome which encode essential genes required to produce ribosomes. DNA double-stranded breaks (DSBs) generated within rDNA genes elicit a unique cellular response involving robust transcriptional silencing and nucleolar reorganization into ‘cap’ structures at the nucleolar periphery. This process is coordinated by the nucleolar scaffolding protein TCOF1, which functions to recruit the DNA repair proteins NBS1 and TOPBP1 that activate the ATM and ATR kinases, resulting in ribosomal RNA (rRNA) transcriptional silencing and nucleolar segregation. However, the DNA damage and repair response at rDNA arrays remains incompletely understood. Here, we investigate the cellular response to rDNA DSBs using proteomics and genetic CRISPR-Cas9 screening. We show that the protein UFMylation pathway and the HUSH complex are important for cell viability and survival in response to rDNA DSBs, and that the E3 UFM1-ligase UFL1 and its heterodimer DDRGK1 are associated with TCOF1 at nucleolar caps. Loss of UFL1 leads to impaired ATM activation, reduced rRNA transcriptional silencing, and an overall reduction in nucleolar segregation. We identified ATM, UNC45A and SMC6 as UFMylated proteins, in which UFMylation may facilitate ATM activation and segregation of damaged rDNA to the nucleolar periphery. Altogether, our findings provide the first evidence for a role for UFMylation in rDNA DSB repair.
2024-09-16 | PXD048568 | Pride
Project description:Comprehensive Model Description
Overview
This model simulates the cellular response to DNA damage under conditions of ATM/ATR deficiency—such as in ataxia telangiectasia—and predicts the potential benefits of drug repositioning with Omaveloxolone and spermidine. It integrates several interconnected modules: DNA damage production and repair, ATM/ATR–p53 signaling, NRF2/KEAP1 regulation, spermidine-induced autophagy (with additional positive modulation of ATR signaling), and downstream p53 effectors involved in cell fate decisions.
1. DNA Damage Production and Repair Module
Basal DNA Damage Production:
DNA damage is continuously generated at a constant rate, representing both endogenous processes and low-level environmental stress. This reaction acts as a source term for the DNA_damage species.
Repair Pathways:
Several mechanisms work to reduce DNA damage:
ATM-Mediated Repair: Active ATM facilitates the direct repair of DNA damage.
ATR-Mediated Repair: Active ATR, in cooperation with CHK1, repairs DNA damage.
NRF2-Mediated Repair: Active NRF2 triggers antioxidant responses that reduce DNA damage.
Autophagy-Mediated Repair: The autophagy pathway, induced by spermidine, repairs DNA damage in a saturable manner (modeled using Michaelis–Menten kinetics). This ensures that even a significant pulse of damage is only partially repaired, leaving a nonzero steady-state level.
2. ATM/ATR–p53 Signaling Module
ATM and ATR Activation:
DNA damage, along with a positive input from TOPBP1, converts inactive ATM and ATR to their active forms. Under ATM/ATR-deficient conditions (as in ataxia telangiectasia), the activation is severely reduced, resulting in higher levels of unrepaired DNA damage.
p53 Activation:
Both ATM_active and ATR_active phosphorylate and activate p53. Active p53 then acts as a central integrator of the DNA damage signal.
Regulatory Proteins:
HDAC4:
HDAC4 cycles between inactive and active states. Its active form can attenuate p53 activity.
TOPBP1:
TOPBP1 is activated in a DNA damage–dependent manner and helps promote ATR activation.
CK2:
CK2 is a constitutively active kinase that supports TOPBP1 activation.
3. NRF2/KEAP1 Module and Omaveloxolone
NRF2 Activation:
DNA damage stimulates the conversion of NRF2_inactive to NRF2_active. Active NRF2 enhances antioxidant defenses and promotes DNA repair.
KEAP1-Mediated Degradation:
Under normal conditions, KEAP1 targets NRF2_inactive for degradation, keeping NRF2 activity low.
Modulation by Omaveloxolone:
Omaveloxolone is modeled as a constant (boundary) species. It inhibits KEAP1’s activity, thereby reducing NRF2 degradation. This shifts the balance in favor of increased NRF2_active, which boosts the cell’s antioxidant and repair responses.
4. Spermidine-Induced Autophagy and Positive Modulation of ATR Signaling
Spermidine is modeled as a constant species and has two major effects:
Induction of Autophagy:
Spermidine promotes the conversion of Autophagy_inactive to Autophagy_active. The active autophagy machinery repairs DNA damage using a saturable (Michaelis–Menten) mechanism. This pathway helps clear DNA damage but is limited by its maximum capacity.
Enhancement of ATR Signaling:
Spermidine also positively modulates ATR signaling by enhancing the CK2-mediated activation of TOPBP1. In the model, the effective rate of TOPBP1 activation is increased proportionally to the concentration of spermidine. This is represented by a modulation factor, where the effective rate constant for TOPBP1 activation is multiplied by a term that increases with spermidine concentration. The boosted TOPBP1_active level in turn enhances ATR activation, partially compensating for the loss of ATM/ATR function.
5. Downstream p53 Targets: Cell Fate Decisions
Activated p53 regulates cell fate by inducing the production of several downstream effectors:
p21:
p21 is produced in response to p53 activation and is associated with cell cycle arrest and senescence. Its production is balanced by a degradation reaction.
BAX and PUMA:
Both BAX and PUMA are pro-apoptotic proteins. They are produced in proportion to p53_active and are subsequently degraded. Their relative levels help determine whether the cell will undergo apoptosis.
6. Reaction Kinetics (Qualitative Overview)
Mass-Action and Michaelis–Menten Kinetics:
Most reactions in the model are governed by mass-action kinetics. For example, activation and deactivation reactions (such as ATM activation by DNA damage or p53 activation by ATM/ATR) follow simple proportional relationships.
Saturable Repair Processes:
The autophagy-mediated DNA repair reaction uses Michaelis–Menten kinetics to reflect a saturation effect; when DNA damage is high, the repair rate approaches a maximum value, ensuring that not all damage is cleared immediately.
Modulatory Effects:
Spermidine increases the effective rate of TOPBP1 activation (and hence ATR activation) via a modulation term that is proportional to its concentration. Omaveloxolone reduces the degradation rate of NRF2 by inhibiting KEAP1.
Biological Implications and Model Utility
ATM/ATR Deficiency:
In ataxia telangiectasia, ATM/ATR functions are diminished, leading to increased DNA damage. The model simulates this scenario and examines how alternative pathways (NRF2-mediated repair, autophagy, and enhanced ATR signaling via spermidine) help mitigate the damage.
Drug Repositioning Predictions:
The model can be used to predict the outcomes of repositioning drugs:
Omaveloxolone is expected to stabilize NRF2, enhancing antioxidant defenses and reducing DNA damage.
Spermidine acts dually by promoting autophagy (with a saturable repair capacity) and by boosting ATR signaling via TOPBP1 activation.
Cell Fate Outcomes:
The downstream p53 targets (p21, BAX, and PUMA) serve as markers for cell cycle arrest/senescence and apoptosis. By simulating the dynamics of these proteins, the model can predict whether a cell is likely to survive, arrest its cycle, or undergo apoptosis based on the integrated response to DNA damage and drug interventions.
Conclusion
This detailed and comprehensive model integrates multiple layers of the cellular response to DNA damage in ATM/ATR-deficient conditions. It includes upstream damage sensing, multiple repair pathways (including direct kinase-mediated repair, antioxidant responses via NRF2, and autophagy-mediated repair), and downstream effectors that determine cell fate. Omaveloxolone and spermidine are modeled to specifically modulate the NRF2/KEAP1 pathway and ATR signaling (via TOPBP1 activation), respectively. This framework provides a robust platform for exploring drug repositioning strategies and predicting cellular outcomes in diseases such as ataxia telangiectasia.
2025-11-25 | MODEL2503190002 | BioModels
Project description:In mammals and several other taxa, the ability of males to cope with the limited synapsis of the X and Y chromosomes during prophase I of meiosis relies on the process of meiotic sex chromosome inactivation (MSCI). Components of the somatic DNA damage response machinery, including ATR, TOPBP1, MDC1 and BRCA1 play key roles in MSCI, although how they establish XY silencing remains incompletely understood. In particular, it remains unclear how DDR factors coordinate XY silencing with DNA repair, chromosome synapsis and the formation of the sex body, a distinct phase-separated sub-nuclear structure formed during prophase I to house the unsynapsed XY bivalent. Here we report a mutant mouse (Topbp1B5/B5), harboring mutations in the BRCT5 domain of Topbp1, that shows impaired XY silencing but grossly normal sex body formation. While Topbp1B5/B5 mice are viable, without detectable somatic defects, males are completely infertile. Distinct from mice lacking ATR or TOPBP1 specifically during meiosis, Topbp1B5/B5 males exhibit normal chromosome synapsis and canonical markers of DNA repair in early prophase I. ATR signaling is mostly intact in Topbp1B5/B5 spermatocytes, although specific ATR-dependent events are disrupted, including localization of the RNA:DNA helicase Senataxin to chromatin loops of the XY. Strikingly, while Topbp1B5/B5 spermatocytes are able to initiate MSCI the completion of gene silencing is defective, with a subset of X chromosome genes displaying distinct patterns of transcriptional deregulation. These findings suggest a non-canonical role for the ATR-TOPBP1 signaling axis in XY silencing dynamics at advanced stages in pachynema. This is the first DDR mutant that separates XY silencing from sex body formation, as well as TOPBP1’s role in spermatogenesis from its roles in organismal viability.
2024-02-09 | PXD042199 | Pride
Project description:To investigate the regulation of protein phosphorylation during the DNA damage response and the role of ATR kinase activity, we performed a comparative phosphoproteomic analysis of HeLa cells treated with etoposide alone or in combination with the ATR inhibitor VE821. Etoposide is a topoisomerase II inhibitor that induces DNA double-strand breaks, activating the DNA damage signaling cascade. We focused particularly on phosphorylation events associated with Treacle (TCOF1), a nucleolar protein implicated in the regulation of rDNA transcription and nucleolar DNA damage response. Quantitative mass spectrometry-based phosphoproteomics was used to identify and quantify global phosphorylation changes under both treatment conditions. This dataset enables the identification of ATR-dependent phosphorylation sites and may provide insights into the molecular mechanisms underlying nucleolar stress responses and the coordination of DNA repair processes.
2026-05-25 | PXD067042 | Pride
Project description:DNA Double Strand Breaks (DSBs) are harmful lesions that require rapid detection and repair in order to avoid toxic genomic rearrangements. DSBs elicit the so called DNA Damage Response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA Protein Kinase (DNAPK), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA cell line, expressing the AsiSI restriction enzyme, we have investigated the role of ATM and DNAPK in several aspects of the DDR upon induction of multiple clean DSBs throughout the human genome. High resolution mapping revealed that they are activated and spread in cis on a confined region surrounding all DSBs, independently of the pathway used for repair. However, a thorough analysis of repair kinetics, H2AX domain establishment and H2AX foci structure and dynamics revealed non overlapping functions for the two kinases once recruited at DSBs. Our results suggest that ATM is not solely acting on chromatin marks but also on chromatin organisation in order to ensure repair accuracy and survival.
2015-09-30 | E-MTAB-2992 | biostudies-arrayexpress
Project description:Next generation sequencing of neuroblastoma (NB) tumors have revealed frequent somatic and germline genetic alterations in genes encoding proteins involved in DNA damage response (DDR) pathways. Despite being well-studied in many adult cancers, roles for DDR disruption in pediatric solid tumors have not been fully elucidated. To address this, patient-relevant loss-of-function mutations in DDR pathway components including Brca2, Atm, and Palb2 were incorporated into an established zebrafish MYCN transgenic model (Tg(dbh:EGFP-MYCN)). These mutations were found to enhance NB formation and metastasis in vivo, and result in upregulation of proliferation, cell cycle checkpoint and DNA damage repair transcriptional signatures, revealing novel molecular vulnerabilities in DDR-deficient NB. Zebrafish DDR-deficient NB and human NB cells with DDR protein knock-down were sensitive to the poly(ADP-ribose)-polymerase (PARP) inhibitor olaparib, and this effect was further enhanced by inhibition of the ataxia telangiectasia and rad3 related (ATR) kinase. Altogether, our data supports a functional role for DDR-deficiency in NB in vivo and therapeutic potential for combination PARP + ATR inhibition in NB patients with alterations in DDR genes.
2025-02-10 | GSE283976 | GEO
Project description:The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3-kinase-related protein kinases (PIKKs): ATM, ATR and DNA-PK. ATM is missing or inactivated in the genome instability syndrome, ataxia-telangiectasia (A-T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild-type and A-T cells treated with the double-strand break-inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine-tuned dynamics between the PIKKs following genotoxic stress, such as DNA-PK-dependent attenuation of ATM. In A-T cells, partial compensation for ATM absence was provided by ATR and DNA-PK, with distinct roles and kinetics. Our results highlight the intricate relationships between these PIKKs in regulating the DDR.
2020-11-25 | PXD020884 | Pride
Project description:Jaiswal2017 - Cell cycle arrest
This model is described in the article:
ATM/Wip1 activities at
chromatin control Plk1 re-activation to determine G2 checkpoint
duration.
Jaiswal H, Benada J, Müllers E,
Akopyan K, Burdova K, Koolmeister T, Helleday T, Medema RH,
Macurek L, Lindqvist A.
EMBO J. 2017 Jul; 36(14):
2161-2176
Abstract:
After DNA damage, the cell cycle is arrested to avoid
propagation of mutations. Arrest in G2 phase is initiated by
ATM-/ATR-dependent signaling that inhibits mitosis-promoting
kinases such as Plk1. At the same time, Plk1 can counteract
ATR-dependent signaling and is required for eventual resumption
of the cell cycle. However, what determines when Plk1 activity
can resume remains unclear. Here, we use FRET-based reporters
to show that a global spread of ATM activity on chromatin and
phosphorylation of ATM targets including KAP1 control Plk1
re-activation. These phosphorylations are rapidly counteracted
by the chromatin-bound phosphatase Wip1, allowing cell cycle
restart despite persistent ATM activity present at DNA lesions.
Combining experimental data and mathematical modeling, we
propose a model for how the minimal duration of cell cycle
arrest is controlled. Our model shows how cell cycle restart
can occur before completion of DNA repair and suggests a
mechanism for checkpoint adaptation in human cells.
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2024-09-02 | BIOMD0000000641 | BioModels