Biology subjects

Bartek, J.

Publications and source records attributed to Bartek, J..

11 recordsLinked to original sources

bioRxiv2025

Ubiquitin-dependent degradation of p21Waf1/Cip1 is mediated by AMBRA1 to limit DNA replication stress

The cell cycle is a fundamental process that orchestrates the events that lead to cell replication and division. AMBRA1 is a scaffold factor that interacts with the E3 ubiquitin ligase CRL4-DDB1 complex to regulate the stability of cyclin D-type proteins, key regulators of the G1/S phase transition. However, how cells complete S-phase and coordinate the speed of DNA synthesis is still to be fully elucidated. Here we demonstrate that AMBRA1 affects the turnover of p21Waf1/Cip1 and p27Kip1, by coupling the CRL4-DDB1 complex directly to these proteins. In the absence of AMBRA1, the increased stability of p21Waf1/Cip1, rather than p27Kip1, resulted in the accumulation of replication stress by leaving under-replicated DNA during the S phase. We also found that AMBRA1-depleted cells with consequent high p21Waf1/Cip1 are sensitive to the inhibition of lagging-strand DNA synthesis. Of clinical relevance, low levels of AMBRA1 in Sonic-Hedgehog medulloblastoma correlate with a worse prognosis, suggesting that AMBRA1 expression levels could be an informative criterion for patient stratification and treatment.

molecular biology↗

bioRxiv2025

Spatial mapping of DNA synthesis reveals dynamics and geometry of human replication nanostructures

DNA replication is essential to life and ensures the accurate transmission of genetic information, which is significantly disturbed during cancer development and chemotherapy. While DNA replication is tightly controlled in time and space, methods to visualise and quantify replication dynamics within 3D human cells are lacking. Here, we introduce 3D-Spatial Assay for Replication Kinetics (3D-SPARK), an approach enabling nanoscale analysis of DNA synthesis dynamics in situ. 3D-SPARK integrates optimised nucleotide analogue pulse labelling with super-resolution microscopy to detect, classify, and quantify replication nanostructures in single cells. By combining immunofluorescence techniques with click chemistry-based nascent DNA labelling and transfection of fluorescent nucleotide derivatives, we map multi-colour DNA synthesis events in relation to established replication proteins, local RNA-protein condensates or large subnuclear domains. We demonstrate quantitative changes in size, relative abundance and spatial arrangement of nanoscale DNA synthesis events upon chemotherapeutic treatment, CDC6 oncogene expression and loss of chromatin organiser RIF1. The flexibility, precision and modular design of 3D-SPARK helps bridging the gap between spatial cell biology, genomics, and 2D fibre-based replication studies in health and disease.

cell biology↗

bioRxiv2024

Glutamine-addiction in cisplatin resistant cancer cells is mediated by SLC7A11 and can be targeted with asparaginase therapy

Linking disease phenotypes with molecular targets is key to the rational design of treatment interventions. Resistance to the chemotherapeutic cisplatin is one of the major factors limiting the clinical utility of this therapy, which is central to the treatment of a variety of solid malignancies. In this study, we couple the upregulation of a chemoresistant factor, the glutamate-cystine antiporter SLC7A11, with the addiction of cisplatin-resistant cancer cells to extracellular glutamine. In doing so, we thus provide a putative biomarker for this acquired metabolic dependency of chemoresistance. Subsequently, we evaluate various therapeutic strategies to selectively kill SLC7A11high cisplatin-resistant cancer cells, identifying cross-resistance to ferroptosis-inducing compounds and hypersensitivity to glutaminase inhibitor CB-839. We identify enzymatic depletion of extracellular glutamine using the long-standing anti-leukemic therapy asparaginase (ASNase), which possesses glutaminase activity, as a potential approach, and show this can be successfully combined with cisplatin in cell models. In summary, this study mechanistically links an acquired metabolic dependency of chemoresistant cancer cells with a putative biomarker and provides a potentially actionable strategy to target these drug resistant cells warranting further investigation.

cancer biology↗

bioRxiv2023

RNA Polymerase II-mediated transcription is required for repair of ribosomal DNA breaks in nucleolar caps, guarding against genomic instability

Ribosomal DNA (rDNA) double-strand breaks (DSBs) threaten genome integrity due to the repetitive and transcriptionally active nature of rDNA. The nucleolus, while central to ribosome biogenesis, also functions as stress sensor. Here, we identify a transcription-dependent mechanism in which RNA polymerase II (RNAPII) is essential for homologous recombination (HR) repair of rDNA DSBs. Using CRISPR-induced breaks, high-resolution imaging, and transcriptional inhibition, we show that RNAPII activity drives the formation of nucleolar repair caps. Mechanistically, CtIP promotes RNAPII recruitment and H3K36 trimethylation at rDNA lesions, facilitating HR. Disruption of this RNAPII-CtIP-H3K36me3 axis impairs cap formation and repair, leading to persistent damage. RNAPII inhibition exacerbates genome instability and synergizes with rDNA breaks to induce cancer cell death, without acutely impairing ribosome function. These findings uncover a co-transcriptional mechanism of rDNA repair and highlight RNAPII-mediated chromatin remodeling and spatial reorganization as key to nucleolar genome maintenance and potential targets for cancer therapy. TeaserNascent RNA synthesis by RNAPII safeguards ribosomal DNA integrity to avoid genomic instability in human cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=137 HEIGHT=200 SRC="FIGDIR/small/563274v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@8e9b03org.highwire.dtl.DTLVardef@63116eorg.highwire.dtl.DTLVardef@d583dorg.highwire.dtl.DTLVardef@1c2eab_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Topological stress triggers difficult-to-repair DNA lesions in ribosomal DNA with ensuing formation of PML-nucleolar compartment

PML, a multifunctional protein, is crucial for forming PML-nuclear bodies involved in stress responses. Under specific conditions, PML associates with nucleolar caps formed after RNA polymerase I (RNAPI) inhibition, leading to PML-nucleolar associations (PNAs). This study investigates PNAs-inducing stimuli by exposing cells to various genotoxic stresses. We found that the most potent inducers of PNAs introduced topological stress and inhibited RNAPI. Doxorubicin, the most effective compound, induced double-strand breaks (DSBs) in the rDNA locus. PNAs co-localized with damaged rDNA, segregating it from active nucleoli. Cleaving the rDNA locus with I-PpoI confirmed rDNA damage as a genuine stimulus for PNAs. Inhibition of ATM, ATR kinases, and RAD51 reduced I-PpoI-induced PNAs, highlighting the importance of ATM/ATR-dependent nucleolar cap formation and homologous recombination (HR) in their triggering. I-PpoI-induced PNAs co-localized with rDNA DSBs positive for RPA32-pS33 but deficient in RAD51, indicating resected DNA unable to complete HR repair. Our findings suggest that PNAs form in response to persistent rDNA damage within the nucleolar cap, highlighting the interplay between PML/PNAs and rDNA alterations due to topological stress, RNAPI inhibition, and rDNA DSBs destined for HR. Cells with persistent PNAs undergo senescence, suggesting PNAs help avoid rDNA instability, with implications for tumorigenesis and aging.

cell biology↗

bioRxiv2022

CtIP-dependent nascent RNA expression flanking DNA breaks guides the choice of DNA repair pathway

The RNA world is changing our views about sensing and resolution of DNA damage. Here, we developed single-molecule DNA/RNA analysis approaches to visualize how nascent RNA facilitates the repair of DNA double-strand breaks (DSBs). RNA polymerase II (RNAPII) is crucial for DSB resolution in human cells. DSB-flanking, RNAPII-generated nascent RNA forms RNA:DNA hybrids, guiding the upstream DNA repair steps towards favouring the error-free Homologous Recombination (HR) pathway over Non-Homologous End Joining. Specific RNAPII inhibitor, THZ1, impairs recruitment of essential HR proteins to DSBs, implicating nascent RNA in DNA end resection, initiation and execution of HR repair. We further propose that resection factor CtIP interacts with and re-activates RNAPII when paused by the RNA:DNA hybrids, collectively promoting faithful repair of chromosome breaks to maintain genomic integrity.

molecular biology↗

bioRxiv2021

The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin

Summary paragraphPoly-(ADP-ribose) polymerase inhibitors (PARPi) elicit anti-tumour activity in homologous recombination defective cancers by promoting cytotoxic, chromatin-bound, "trapped" PARP1. How cells process trapped PARP1 remains unclear. By exploiting wild-type or trapping-resistant PARP1 transgenes combined with either a rapid immunoprecipitation mass-spectrometry of endogenous proteins (RIME)-based approach, or PARP1 Apex2-proximity labelling linked to mass-spectrometry, we generated proteomic profiles of trapped and non-trapped PARP1 complexes. This combined approach identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase (aka VCP). Subsequent experiments demonstrated that upon trapping, PARP1 is SUMOylated by the SUMO-ligase PIAS4 and subsequently ubiquitinated by the SUMO-targeted E3-ubiquitin ligase, RNF4, events that promote p97 recruitment and p97 ATPase-mediated removal of trapped-PARP1 from chromatin. Consistent with this, small molecule p97 complex inhibitors, including a metabolite of the clinically-used drug disulfiram (CuET) that acts as a p97 sequestration agent, prolong PARP1 trapping and thus enhance PARPi-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Taken together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 from chromatin and the response of homologous recombination defective tumour cells to PARPi.

molecular biology↗

bioRxiv2021

Loss of Nuclear DNA ligase III Can Revert PARP Inhibitor Resistance in BRCA1-deficient Cells by Increasing DNA Replication Stress

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

cancer biology↗

bioRxiv2020

Genomic instability is an early event driving chromatin reorganization and escape from oncogene-induced senescence

Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not timely removed via immune surveillance, senescent cells will also present a detrimental side. Although this has mostly been attributed to the senescence-associated-secretory-phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state represents another unfavorable outcome. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion, which harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and for driving cell cycle re-entry and malignant transformation. In summary, we now provide strong evidence in support of genomic instability underlying "escape" from oncogene-induced senescence. HIGHLIGHTSO_LIOncogene driven error-prone repair produces early genetic lesions allowing escape from senescence C_LIO_LICells escaping oncogene-induced senescence display mutational signatures observed in cancer patients C_LIO_LIA single recurrent inversion harboring a circadian TF gene suffices for bypassing oncogene-induced senescence C_LIO_LIChromatin loop and compartment remodeling support the "escape" transcriptional program C_LI

cell biology↗

bioRxiv2020

A chemical screen identifies a link between lipid metabolism and mRNA translation

mRNA translation is one of the most energy-demanding processes for living cells, alterations of which have been frequently documented in human disease. Using recently developed technologies that enable image-based quantitation of overall translation levels, we here conducted a chemical screen to evaluate how medically approved drugs, as well as drugs that are currently under development, influence overall translation levels. Consistent with current knowledge, inhibitors of the mTOR signaling pathway were the most represented class among translation suppresors. In addition, we identified that inhibitors of sphingosine kinases (SPHKs) also reduce mRNA translation levels independently of mTOR. Mechanistically this is explained by an effect of the compounds on the membranes of the endoplasmic reticulum, which activates the integrated stress response (ISR). Accordingly, the impact of SPHK inhibitors on translation is alleviated by the concomitant inhibition of ISR kinases. On the other hand, and despite the large number of molecules tested, our study failed to identify chemicals capable of substantially increasing mRNA translation, raising doubts on to what extent translation can be supra-physiologically stimulated in mammalian cells. In summary, our study provides the first comprehensive characterization of the effect of known drugs on protein translation and has helped to unravel a new link between lipid metabolism and mRNA translation in human cells.

molecular biology↗

bioRxiv2017

Mutational signatures reveal the role of RAD52 in p53-independent p21 driven genomic instability

BackgroundGenomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential to design appropriate therapeutic strategies. In a recent study we reported an unexpected oncogenic property of p21WAF1/Cip1 showing that its chronic expression, in a p53-deficient environment, causes genomic instability by deregulating the replication licensing machinery.\n\nResultsExtending on this work we now demonstrate that p21WAF1/Cip1 can further fuel genomic instability by suppressing the repair capacity of low and high fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we found at the mechanistic level that the DSBs were repaired by Rad52-dependent Break-Induced Replication (BIR) and Single-Strand Annealing (SSA). Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route was deficient. Surprisingly, Rad52 was activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation.\n\nConclusionsOur results signify the importance of mutational signatures as guides to disclose the \"repair history\" leading to genomic instability. In this vein, following this approach we unveiled how chronic p21WAF1/Cip1 expression rewires the repair process, identifying Rad52 as a source of genomic instability and a candidate therapeutic target.

cancer biology↗