bims-ginsta Biomed News
on Genome instability
Issue of 2025–09–14
35 papers selected by
Jinrong Hu, National University of Singapore
  1. Combinatorial BMP4 and activin direct the choice between alternate routes to endoderm in a stem cell model of human gastrulation.
  2. Real-time visualization of reconstituted transcription reveals RNAPII activation mechanisms at single promoters.
  3. Temporal control of human DNA replication licensing by CDK4/6-RB signalling and chemical genetics.
  4. The Arp2/3 complex maintains gut epithelial integrity under mechanical challenge.
  5. Hbo1 and Msl complexes preserve differential compaction and H3K27me3 marking of active and inactive X chromosomes during mitosis.
  6. Tension-sensitive LINC-RhoA signaling prevents chromatin bridge breakage in cytokinesis.
  7. Digital reconstruction of full embryos during early mouse organogenesis.
  8. Deep visual proteomics reveals an in vivo-like phenotype of orthotopically transplanted human colon organoids.
  9. Multiple human enhancer RNAs contain long translated open reading frames.
  10. Morphogenesis and regeneration share a conserved core transition cell state program that controls lung epithelial cell fate.
  11. Human gastroids to model regional patterning in early stomach development.
  12. Durotaxis is a driver and potential therapeutic target in lung fibrosis and metastatic pancreatic cancer.
  13. m6A and the NEXT complex direct Xist RNA turnover and X-inactivation dynamics.
  14. Aurora A regulates the material property of spindle poles to orchestrate nuclear organization at mitotic exit.
  15. An alternative route for β-hydroxybutyrate metabolism supports cytosolic acetyl-CoA synthesis in cancer cells.
  16. Amplifying antigen-induced cellular responses with proximity labelling.
  17. α-Synuclein aggregates inhibit ESCRT-III through sequestration and collateral degradation.
  18. Protection against ferroptosis through maintaining homeostasis of docosahexaenoate-containing phospholipids.
  19. The DNA replication machinery transmits dual signals to prevent unscheduled licensing and execution of centrosome duplication.
  20. Nuclear compression-mediated DNA damage drives ATR-dependent Lamin expression and mouse ESC differentiation.
  21. Energy deficiency selects crowded live epithelial cells for extrusion.
  22. A snapshot of proteostasis in human oocytes.
  23. Mitochondria-associated condensates maintain mitochondrial homeostasis and promote lifespan.
  24. Dynamic association of H3K36me3 with pericentromeric heterochromatin regulates its replication time.
  25. Matrix directs trophoblast differentiation in a bioprinted organoid model of early placental development.
  26. Dynamic regulation of the oxidative stress response by the E3 ligase TRIP12.
  27. A novel DNA repair-independent role for Gen nuclease in promoting unscheduled polyploidy cell proliferation.
  28. Structural basis for Rad54- and Hed1-mediated regulation of Rad51 during the transition from mitotic to meiotic recombination.
  29. Tubulin hyperacetylation drives HMGB1 nuclear exit via the ROS-PARP1 axis leading to rotenone-induced G2/M Arrest.
  30. HT SpaceM: A high-throughput and reproducible method for small-molecule single-cell metabolomics.
  31. Emergent depth-mechanosensing of epithelial collectives regulates cell clustering and dispersal on layered matrices.
  32. DNA polymerase α-primase can function as a translesion DNA polymerase.
  33. Genome-wide mapping of RNA-protein associations through sequencing.
  34. SEPTIN9 locally activates the RhoGEF ARHGEF18 to promote early stages of mitochondrial fission.
  35. A rapid imaging-based screen for induced-proximity degraders identifies a potent degrader of oncoprotein SKP2.
  1. Dev Cell. 2025 Sep 09. pii: S1534-5807(25)00529-5. [Epub ahead of print]
    Combinatorial BMP4 and activin direct the choice between alternate routes to endoderm in a stem cell model of human gastrulation.
    Oliver C K Inge, Elias Copin, Jake Cornwall-Scoones, Borzo Gharibi, Irene Rodriguez-Hernandez, Pablo Soro-Barrio, Molly Strom, Probir Chakravarty, James Briscoe, Silvia D M Santos.
      Lineage specification requires accurate interpretation of multiple signaling cues. However, how combinatorial signaling histories influence fate outcomes remains unclear. We combined single-cell transcriptomics, live-cell imaging, and mathematical modeling to explore how activin and bone morphogenetic protein 4 (BMP4) guide fate specification during human gastrulation. We see that these signals interact both synergistically and antagonistically to drive fate decisions. We find that definitive endoderm arises from lineage convergence: a direct route from pluripotency and an indirect route via a mesoderm progenitor state. Cells pass through temporal windows of signaling competency, and the relative concentration of activin and BMP4 dictates the trajectory choice. The efficiency between routes is underpinned by a dual role of BMP4 in inducing mesoderm genes while promoting pluripotency exit. This work underscores that the combination of signals a cell is exposed to not only directs its final fate but also the developmental route taken, suggesting lineage convergence enhances robustness in fate specification.
    Keywords:  BMP4; activin; combinatorial signaling; endoderm; fate specification; hESC; human gastrulation; modeling; scRNA-seq; state transitions
    DOI:  https://doi.org/10.1016/j.devcel.2025.08.009
  2. Cell Rep. 2025 Sep 05. pii: S2211-1247(25)01022-8. [Epub ahead of print]44(9): 116251
    Real-time visualization of reconstituted transcription reveals RNAPII activation mechanisms at single promoters.
    Megan Palacio, Dylan J Taatjes.
      RNA polymerase II (RNAPII) is regulated by sequence-specific transcription factors (TFs) and the pre-initiation complex (PIC): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and Mediator. TFs, Mediator, and RNAPII contain intrinsically disordered regions (IDRs) and form phase-separated condensates, but how IDRs control RNAPII function remains poorly understood. Using purified PIC factors, we developed a real-time in vitro fluorescence transcription (RIFT) assay for second-by-second visualization of transcription at hundreds of promoters simultaneously. Our results establish IDRs as essential for rapid RNAPII activation, without condensate formation. For example, HSF1 condensates and single molecules function identically, whereas MED1-IDR can functionally replace HSF1 but activates RNAPII with slower kinetics. Through their IDRs, Mediator and TFs rapidly and synergistically activate RNAPII bursting and re-initiation, and surprisingly, Mediator drives TF-promoter recruitment without TF-DNA binding. Importantly, RIFT directly addresses questions largely intractable with cell-based methods, yielding mechanistic insights about condensates, IDRs, enhancer-promoter communication, and RNAPII bursting that complement live-cell imaging data.
    Keywords:  CP: Molecular biology; HSF1; Mediator; PIC scaffold; RNA polymerase II; TFIID; burst size; bursting; condensates; fluorescence microscopy; intrinsically disordered regions; phase separation; re-initiation; smTIRF; transcription; transcription factor
    DOI:  https://doi.org/10.1016/j.celrep.2025.116251
  3. Nat Commun. 2025 Sep 12. 16(1): 8268
    Temporal control of human DNA replication licensing by CDK4/6-RB signalling and chemical genetics.
    Anastasia Sosenko Piscitello, Ann-Sofie Nilsson, Michael Hawgood, Abid H Sayyid, Vasilis S Dionellis, Giovanni Giglio, Bruno Urién, Pratikiran Bajgain, Sotirios G Ntallis, Jiri Bartek, Thanos D Halazonetis, Bennie Lemmens.
      Cyclin-dependent kinases (CDKs) coordinate DNA replication and cell division, and play key roles in tissue homeostasis, genome stability and cancer development. The first step in replication is origin licensing, when minichromosome maintenance (MCM) helicases are loaded onto DNA by CDC6, CDT1 and the origin recognition complex (ORC). In yeast, origin licensing starts when CDK activity plummets in G1 phase, reinforcing the view that CDKs inhibit licensing. Here we show that, in human cells, CDK4/6 activity promotes origin licensing. By combining rapid protein degradation and time-resolved EdU-sequencing, we find that CDK4/6 activity acts epistatically to CDC6 and CDT1 in G1 phase and counteracts RB pocket proteins to promote origin licensing. Therapeutic CDK4/6 inhibitors block MCM and ORC6 loading, which we exploit to trigger mitosis with unreplicated DNA in p53-deficient cells. The CDK4/6-RB axis thus links replication licensing to proliferation, which has implications for human cell fate control and cancer therapy design.
    DOI:  https://doi.org/10.1038/s41467-025-63669-8
  4. Curr Biol. 2025 Sep 09. pii: S0960-9822(25)01092-9. [Epub ahead of print]
    The Arp2/3 complex maintains gut epithelial integrity under mechanical challenge.
    Louisiane Perrin, Venkata Ram Gannavarapu, Carlos Pérez-González, Claudia Rivera, Stéphanie Descroix, Ana-Maria Lennon-Duménil, Yohanns Bellaïche, Denis Krndija, Danijela Matic Vignjevic.
      Epithelia are specialized and selective tissue barriers that separate the organism's interior from the external environment. Among adult tissues, the gut epithelium must withstand microbial and biochemical insults but also mechanical stresses imposed by luminal contents and gastrointestinal motility. In addition, the continuous renewal of the intestinal epithelium creates tension1,2 that must be withstood by cell-cell junctions and the actomyosin cytoskeleton to preserve barrier integrity.3,4,5,6,7,8,9,10,11,12 Despite these continuous challenges, the intestinal epithelium maintains a robust barrier function, though the underlying mechanisms remain poorly understood. Among the multiple actin regulators present at cell-cell junctions, the Arp2/3 complex acts as a mechanosensitive nucleator of F-actin at adherens junctions13 and is critical for maintaining cell adhesions in vitro14,15,16,17 and in C. elegans intestinal18 and Drosophila notum19 epithelial cells. Here, we identify the actin nucleator Arp2/3 complex as a critical regulator of intestinal epithelial integrity under mechanical stress. Using a gut epithelium-specific, inducible Arpc4 knockout mouse model, we show that Arp2/3 loss of function leads to increased intestinal permeability, epithelial fracturing, and, ultimately, lethality. Arp2/3 depletion disrupts tight junction protein localization, compromising epithelial stability and making it prone to functional failure. Using ex vivo cultured intestinal slices and intestinal epithelial organoids, we found that these functional defects require mechanical challenge and elevated actomyosin contractility to manifest. Together, our findings establish Arp2/3 as a key regulator of intestinal epithelial homeostasis, ensuring tight junction stability, thus highlighting potential therapeutic targets for disorders associated with barrier dysfunction and inflammation.
    Keywords:  cell-cell junctions; epithelial integrity; intestine; mechanical challenge
    DOI:  https://doi.org/10.1016/j.cub.2025.08.026
  5. Nat Cell Biol. 2025 Sep;27(9): 1482-1495
    Hbo1 and Msl complexes preserve differential compaction and H3K27me3 marking of active and inactive X chromosomes during mitosis.
    Dounia Djeghloul, Sherry Cheriyamkunnel, Bhavik Patel, Holger Kramer, Alex Montoya, Karen E Brown, Chad Whilding, Tatyana B Nesterova, Guifeng Wei, Neil Brockdorff, Iga Grządzielewska, Remzi Karayol, Asifa Akhtar, Matthias Merkenschlager, Amanda G Fisher.
      In mammals, chromosome-wide regulatory mechanisms ensure a balance of X-linked gene dosage between males (XY) and females (XX). In female cells, expression of genes from one of the two X chromosomes is curtailed, with selective accumulation of Xist-RNA, Xist-associated proteins, specific histone modifications (for example, H3K27me3) and Barr body formation observed throughout interphase. Here we show, using chromosome flow-sorting, that during mitosis, Xist-associated proteins dissociate from inactive X (Xi) chromosomes, while high levels of H3K27me3 and increased compaction of the Xi relative to active X (Xa), are retained. Proteomic comparison of mitotic Xi and Xa revealed that components of Hbo1 and Msl/Mof histone acetyltransferase complexes are significantly enriched on Xa as compared to Xi and autosomes. By contrast, inhibitors of histone acetylation co-enrich with Xi. Furthermore, inhibition of Hbo1 or deletion of Msl/Mof components functionally abolishes mitotic differences in H3K27me3 marking and chromosome compaction. These data uncover critical roles for acetylation pathways in preserving X chromosome properties during mitosis.
    DOI:  https://doi.org/10.1038/s41556-025-01748-0
  6. EMBO J. 2025 Sep 09.
    Tension-sensitive LINC-RhoA signaling prevents chromatin bridge breakage in cytokinesis.
    Sofia Balafouti, Maria Kabouraki, George Zachos, Eleni Petsalaki.
      In the presence of chromatin bridges in cytokinesis, human cells retain actin-rich structures (actin patches) at the base of the intercellular canal to prevent chromosome breakage. Here, we show that daughter nuclei connected by chromatin bridges are under mechanical tension that requires interaction of the nuclear membrane Sun1/2-Nesprin-2 Linker of Nucleoskeleton and Cytoskeleton (LINC) complex with the actin cytoskeleton, and an intact nuclear lamina. This nuclear tension promotes accumulation of Sun1/2-Nesprin-2 proteins at the base of chromatin bridges and local enrichment of the RhoA-activator PDZ RhoGEF through PDZ-binding to cytoplasmic Nesprin-2 spectrin repeats. In turn, PDZ RhoGEF activates the small GTPase RhoA and downstream ROCK-LIMK-Cofilin and mDia1 signaling to generate actin patches and prevent chromatin bridge breakage in cytokinesis. These findings identify a novel mechanosensing mechanism by which chromatin bridges promote remodeling of the actin cytoskeleton, through tension-induced activation of LINC-PDZ RhoGEF-RhoA signaling, to generate actin patches to preserve genome integrity.
    Keywords:  Actin Patches; Chromatin Bridges; LINC; Nesprin-2; RhoA
    DOI:  https://doi.org/10.1038/s44318-025-00565-3
  7. Cell. 2025 08 21. pii: S0092-8674(25)00622-1. [Epub ahead of print]188(17): 4754-4772.e18
    Digital reconstruction of full embryos during early mouse organogenesis.
    Peng Xie, Juan Shen, Yi Yang, Xinrui Wang, Wei Liu, Hailong Cao, Yanying Zheng, Chen Wu, Guangyao Mao, Linjin Chen, Jingjing He, Weiheng Zheng, Zepu Yang, Xiao Zhang, Xu Jiang, Xianfa Yang, Ke Fang, Zhao Zhang, Xin Xue, Xueting Chen, Chaoyi Wang, Xing Liu, Ling Liu, Xuebiao Yao, Naihe Jing, Wei Xie, Jin Liu, Hua Cao, Zhuojuan Luo, Xiaodong Fang, Chengqi Lin.
      Early organogenesis is a crucial stage in embryonic development, characterized by extensive cell fate specification to initiate organ formation but also by a high susceptibility to developmental defects. Here, we profiled 285 serial sections from six E7.5-E8.0 embryos to generate full spatiotemporal transcriptome and signal maps during early organogenesis at single-cell resolution. By developing SEU-3D, we reconstructed digital embryos, enabling investigation of regionalized gene expression in the native spatial context. We established a space-informed gene-cell co-embedding approach, systematically characterized the spatial atlas of endoderm and mesoderm derivatives, and elucidated signaling networks across germ layers and cell types. Furthermore, we characterized a primordium determination zone (PDZ) formed along the anterior embryonic-extraembryonic interface at E7.75, and it revealed that the coordinated signaling communications contribute to the formation of cardiac primordium. Collectively, the high-resolution "digital embryo" provides significant insights into early organogenesis and a unique spatial platform for studying development and diseases.
    Keywords:  cross-germ-layer signal communication; digital embryo; early organogenesis; primordium determination zone; single-cell 3D transcriptome; cardiac crescent
    DOI:  https://doi.org/10.1016/j.cell.2025.05.035
  8. Cell Syst. 2025 Sep 10. pii: S2405-4712(25)00229-7. [Epub ahead of print] 101396
    Deep visual proteomics reveals an in vivo-like phenotype of orthotopically transplanted human colon organoids.
    Frederik Post, Annika Hausmann, Sonja Kabatnik, Sophia Steigerwald, Alexandra Brand, Ditte L Clement, Jonathan Skov, Kadi Lõhmussaar, Hjalte L Larsen, Martti Maimets, Theresa L Boye, Gabriela Jez, Toshiro Sato, Casper Steenholdt, Florian Rosenberger, Andreas Mund, Ole H Nielsen, Kim B Jensen, Matthias Mann.
      Intestinal epithelial damage predisposes to disorders like inflammatory bowel disease (IBD), with organoid transplantation emerging as a potential treatment. However, it is not known how well organoids recapitulate in vivo intestinal epithelial cells (IECs). We employed deep visual proteomics (DVP), integrating AI-guided cell classification, laser microdissection, and ultra-high-sensitivity proteomics at the single-cell level to generate an in-depth proteome resource of IECs directly isolated from the human colon and organoids. While in vitro organoids display high proliferation and low functional signatures, xenotransplantation induces a remarkable shift toward an in vivo-like phenotype. We recapitulated this transition by modifying culture conditions. Our data provide a comprehensive spatial proteomics resource and validate xenotransplanted organoids as suitable models for studying human IEC behavior with unprecedented molecular detail and demonstrate their clinical potential for patients with IBD and other intestinal disorders. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  IBD; colon; mass spectrometry; organoids; proteomics; spatial proteomics
    DOI:  https://doi.org/10.1016/j.cels.2025.101396
  9. Genes Dev. 2025 Sep 10.
    Multiple human enhancer RNAs contain long translated open reading frames.
    Pavel A Vlasov, Koichi Ogami, Elizabeth Valenzuela, Risa Karakida Kawaguchi, Marko Jovanovic, James L Manley.
      Enhancer RNAs (eRNAs) are transcribed by RNA polymerase IIduring enhancer activation but are typically rapidly degraded in the nucleus. During states of reduced RNA surveillance, however, eRNAs and other similar "noncoding" RNAs (including, e.g., upstream antisense RNAs) are stabilized, and some are exported to the cytoplasm and can even be found on polysomes. Here, we report unexpectedly that ∼12% of human intergenic eRNAs contain long open reading frames (>300 nt), many of which can be actively translated, as determined by ribosome profiling, and produce proteins that accumulate in cells, as shown by mass spectrometry (MS) data. Focusing on the largest of the encoded proteins, which we designated as eORFs, which can be up to ∼45 kDa, we found, remarkably, that most are highly basic, with pIs >11.5. This unusual chemistry reflects a striking overabundance of arginine residues and occurs despite a relative paucity of lysines. Exogenous expression of the 10 largest eORFs revealed that they accumulate stably in cells as full-length proteins, and most localize to the nucleus and associate with chromatin. Identification of interacting proteins by MS suggested possible roles for these proteins in several nuclear processes. The eORFs studied are well conserved among primates, though they are largely absent from other mammals. Notably, several contain human-specific C-terminal extensions and display properties suggestive of de novo gene birth. In summary, we have discovered that a fraction of human eRNAs can function as mRNAs, revealing a new and unexpected role for these transcripts.
    Keywords:  RNA; enhancers; translation
    DOI:  https://doi.org/10.1101/gad.352944.125
  10. Dev Cell. 2025 Sep 05. pii: S1534-5807(25)00534-9. [Epub ahead of print]
    Morphogenesis and regeneration share a conserved core transition cell state program that controls lung epithelial cell fate.
    Xiangyi Ke, Benjamin van Soldt, Lukas Vlahos, Yizhuo Zhou, Jun Qian, Pasquale Laise, Joel George, Claudia Capdevila, Ian Glass, Kelley Yan, Andrea Califano, Wellington V Cardoso.
      
    DOI:  https://doi.org/10.1016/j.devcel.2025.08.014
  11. Nature. 2025 Sep 10.
    Human gastroids to model regional patterning in early stomach development.
    Xia Li, Feng Lin, Qiqi Cui, Shiyu Sun, Shixin Li, Yue Wang, Yiting Wang, Jianbo Bai, Shiyi Liu, Jia Guo, Yizhao Han, Meiru Zhang, Tie Chang, Yifan Zheng, Jianlin Liu, Longqi Liu, Leyun Wang, Jianping Fu, Xin Liu, Bing Bai, Yue Shao.
      The human stomach features distinct, regionalized functionalities along the anterior-posterior axis1,2. Historically, studies on stomach patterning have used animal models to identify the underlying principles3-7. Recently, human pluripotent stem (hPS)-cell-based gastric organoids for modelling domain-specific development of the fundic and antral epithelium are emerging8-10. However, recapitulating self-organized fundic-antral patterning in early stomach organogenesis remains challenging, presenting a considerable barrier for advancing knowledge of stomach organogenesis. Here we report human gastroids-a self-organized multilineage gastric organoid derived from hPS cells-to model gastric fundic-antral patterning in vitro. Through multi-germ-layer co-development, we generate gastroids that feature an epithelial chamber with bipolar fundic-antral patterning, annexed with neural populations near the fundic domain while enveloped by mesenchymal cells, therefore showing molecular, cellular, structural and anatomical similarity to stomach development in vivo. Non-endodermal cells, especially neural populations, function as a critical signalling centre to instruct fundic-antral patterning in gastroids through WNT-mediated crosstalk. Single-cell transcriptomic profiling and genetic silencing further reveal NR2F2 as a key mediator of fundic-antral patterning in gastroid development. This study reveals a principle for instructing gastric patterning and provides a higher-fidelity platform for advancing knowledge of stomach organogenesis and gastric organoid development.
    DOI:  https://doi.org/10.1038/s41586-025-09508-8
  12. Nat Cell Biol. 2025 Sep;27(9): 1543-1554
    Durotaxis is a driver and potential therapeutic target in lung fibrosis and metastatic pancreatic cancer.
    Taslim A Al-Hilal, Maria-Anna Chrysovergi, Paula E Grasberger, Fei Liu, Vera Auernheimer, Yan Zhou, Zebin Xiao, Mark Anthony Leon-Duque, Alba Santos, Tamanna Islam, Matteo Ligorio, Delphine Sicard, Clemens K Probst, Vladimir Vrbanac, Tejaswini S Reddi, Ludovic Vincent, Cassandra Happe, Edward Chaum, Charles R Yates, Kaveh Daneshvar, Allan C Mullen, David Ting, Eric S White, Raghu Kalluri, Christina M Woo, Ellen Puré, Wolfgang H Goldmann, Jose Luis Alonso, Andrew M Tager, Adam J Engler, Daniel J Tschumperlin, David Lagares.
      Durotaxis, cell migration along stiffness gradients, is linked to embryonic development, tissue repair and disease. Despite solid in vitro evidence, its role in vivo remains largely speculative. Here we demonstrate that durotaxis actively drives disease progression in vivo in mouse models of lung fibrosis and metastatic pancreatic cancer. In lung fibrosis, durotaxis directs fibroblast recruitment to sites of injury, where they undergo mechano-activation into scar-forming myofibroblasts. In pancreatic cancer, stiffening of the tumour microenvironment induces durotaxis of cancer cells, promoting metastatic dissemination. Mechanistically, durotaxis is mediated by focal adhesion kinase (FAK)-paxillin interaction, a mechanosensory module that links stiffness cues to transcriptional programmes via YAP signalling. To probe this genetically, we generated a FAK-FATL994E knock-in mouse, which disrupts FAK-paxillin binding, blocks durotaxis and attenuates disease severity. Pharmacological inhibition of FAK-paxillin interaction with the small molecule JP-153 mimics these effects. Our findings establish durotaxis as a disease mechanism in vivo and support anti-durotactic therapy as a potential strategy for treating fibrosis and cancer.
    DOI:  https://doi.org/10.1038/s41556-025-01697-8
  13. Nat Struct Mol Biol. 2025 Sep 09.
    m6A and the NEXT complex direct Xist RNA turnover and X-inactivation dynamics.
    Guifeng Wei, Heather Coker, Lisa Rodermund, Mafalda Almeida, Holly L Roach, Tatyana B Nesterova, Neil Brockdorff.
      X-chromosome inactivation (XCI) in mammals is orchestrated by the noncoding RNA X-inactive-specific transcript (Xist) that, together with specific interacting proteins, functions in cis to silence an entire X chromosome. Defined sites on Xist RNA carry the N6-methyladenosine (m6A) modification and perturbation of the m6A writer complex has been found to abrogate Xist-mediated gene silencing. However, the relative contribution of m6A and its mechanism of action remain unclear. Here we investigate the role of m6A in XCI by applying rapid degron-mediated depletion of METTL3, the catalytic subunit of the m6A writer complex, an approach that minimizes indirect effects because of transcriptome-wide depletion of m6A. We find that acute loss of METTL3 and m6A accelerates Xist-mediated gene silencing and this correlates with increased levels and stability of Xist transcripts. We show that Xist RNA turnover is mediated by the nuclear exosome targeting complex but is independent of the principal nuclear m6A reader protein YTHDC1. Our findings demonstrate that the primary function of m6A on Xist RNA is to promote Xist RNA turnover, which in turn regulates XCI dynamics.
    DOI:  https://doi.org/10.1038/s41594-025-01663-w
  14. EMBO J. 2025 Sep 12.
    Aurora A regulates the material property of spindle poles to orchestrate nuclear organization at mitotic exit.
    Ashwathi Rajeevan, Vignesh Olakkal, Madhumitha Balakrishnan, Dwaipayan Chakrabarty, François Charon, Daan Noordermeer, Sachin Kotak.
      Animal cells dismantle their nuclear envelope (NE) at the beginning and reconstruct it at the end of mitosis. This process is closely coordinated with spindle pole organization: poles enlarge at mitotic onset and reduce in size as mitosis concludes. The significance of this coordination remains unknown. Here, we demonstrate that Aurora A maintains a pole-localized protein NuMA in a dynamic state during anaphase. Without Aurora A activity, NuMA shifts from a dynamic to a solid state and abnormally accumulates at the poles, causing the segregated chromosome sets to bend around the NuMA-enriched poles. NuMA localization at the poles relies on interactions with dynein/dynactin, its coiled-coil domain, and an intrinsically disordered region (IDR). Mutagenesis experiments revealed that cation-π interactions within IDR are key for NuMA pole localization, while glutamine residues trigger the solid-state transition of NuMA upon Aurora A inhibition. We propose that maintaining the proper material properties of the spindle poles is a key step in choreographing the accurate organization of the nucleus and genome post-mitosis.
    Keywords:  Aurora A; Material Property; NuMA; Nuclear Shape; Spindle Poles
    DOI:  https://doi.org/10.1038/s44318-025-00564-4
  15. Nat Metab. 2025 Sep 08.
    An alternative route for β-hydroxybutyrate metabolism supports cytosolic acetyl-CoA synthesis in cancer cells.
    Faith C Kaluba, Thomas J Rogers, Yu-Jin Jeong, Rachel 'Rae' J House, Althea Waldhart, Kelly H Sokol, Samuel R Daniels, Cameron J Lee, Joseph Longo, Amy Johnson, Vincent J Sartori, Ryan D Sheldon, Russell G Jones, Evan C Lien.
      Cancer cells are exposed to diverse metabolites in the tumour microenvironment that are used to support the synthesis of nucleotides, amino acids and lipids needed for rapid cell proliferation. In some tumours, ketone bodies such as β-hydroxybutyrate (β-OHB), which are elevated in circulation under fasting conditions or low glycemic diets, can serve as an alternative fuel that is metabolized in the mitochondria to provide acetyl-CoA for the tricarboxylic acid (TCA) cycle. Here we identify a non-canonical route for β-OHB metabolism that bypasses the TCA cycle to generate cytosolic acetyl-CoA. We show that in cancer cells that can metabolize ketones, β-OHB-derived acetoacetate in the mitochondria can be shunted into the cytosol, where acetoacetyl-CoA synthetase (AACS) and thiolase convert it into cytosolic acetyl-CoA. This alternative metabolic routing allows β-OHB to avoid oxidation in the mitochondria and to be used as a major source of cytosolic acetyl-CoA, even when other key cytosolic acetyl-CoA precursors such as glucose are available in excess. Finally, we demonstrate that ketone body metabolism, including this alternative AACS-dependent route, can support the growth of mouse KrasG12D; Trp53-/- pancreatic tumours grown orthotopically in the pancreas of male mice, as well as the growth of mouse B16 melanoma tumours in male mice fed a calorie-restricted diet. Together, these data reveal how cancer cells use β-OHB as a major source of cytosolic acetyl-CoA to support cell proliferation and tumour growth.
    DOI:  https://doi.org/10.1038/s42255-025-01366-y
  16. Nature. 2025 Sep 10.
    Amplifying antigen-induced cellular responses with proximity labelling.
    Shuojun Li, Yinghui Men, Zihan Wang, Yingcheng Wu, Hao Sun, Mingyang Yin, Xinrui Fan, Guiyun Deng, Zhicheng Yang, Tiange Yang, Yudian Xiao, Hu Zhou, Guangchuan Wang, Jia Fan, Chenqi Xu, Qiang Gao, Shuo Han.
      Antigen-induced clustering of cell surface receptors, including T cell receptors and Fc receptors, represents a widespread mechanism in cell signalling activation1,2. However, most naturally occurring antigens, such as tumour-associated antigens, stimulate limited receptor clustering and on-target responses owing to insufficient density3-5. Here we repurpose proximity labelling6, a method used to biotinylate and identify spatially proximal proteins, to amplify designed probes as synthetic antigen clusters on the cell surface. We develop an in vivo proximity-labelling technology controlled by either red light or ultrasound to covalently tag fluorescein probes at high density near a target antigen. Using T cell receptors as an example, we demonstrate that the amplified fluorescein effectively clusters and directs a fluorescein-binding bispecific T cell engager to induce enhanced T cell activation and cytotoxicity. Noninvasive, tissue-selective labelling in multiple syngeneic mouse tumour models produces potent immune responses that rapidly eradicate treated tumours. Efficient cell lysis further promotes epitope spreading to induce systemic immunity against untreated distal lesions and immune memory against rechallenge. Thus, proximity-labelling chemistry holds promise as a generalized strategy to manipulate antigen-dependent receptor function and cell states.
    DOI:  https://doi.org/10.1038/s41586-025-09518-6
  17. Mol Cell. 2025 Sep 10. pii: S1097-2765(25)00707-5. [Epub ahead of print]
    α-Synuclein aggregates inhibit ESCRT-III through sequestration and collateral degradation.
    Cole S Sitron, Victoria A Trinkaus, Ana Galesic, Maximilian Garhammer, Patricia Yuste-Checa, Ulrich Dransfeld, Dennis Feigenbutz, Jiuchun Zhang, Larysa Ivashko, Irina Dudanova, J Wade Harper, F Ulrich Hartl.
      α-Synuclein aggregation is a hallmark of Parkinson's disease and related synucleinopathies. Extracellular α-synuclein fibrils enter naive cells via endocytosis, followed by transit into the cytoplasm to seed endogenous α-synuclein aggregation. Intracellular aggregates sequester numerous proteins, including subunits of the endosomal sorting complexes required for transport (ESCRT)-III system for endolysosome membrane repair, but the toxic effects of these events remain poorly understood. Using cellular models and in vitro reconstitution, we found that α-synuclein fibrils interact with a conserved α-helix in ESCRT-III proteins. This interaction sequesters ESCRT-III subunits and triggers their proteasomal destruction in a process of "collateral degradation." These twin mechanisms deplete the available ESCRT-III pool, initiating a toxic feedback loop. The ensuing loss of ESCRT function compromises endolysosome membranes, thereby facilitating escape of aggregate seeds into the cytoplasm, facilitating a "second wave" of templated aggregation and ESCRT-III sequestration. We suggest that collateral degradation and the triggering of self-perpetuating systems are general mechanisms of sequestration-induced proteotoxicity.
    Keywords:  CHMP2B; ESCRT; ESCRT-III; Parkinson’s disease; aggregation; lysosome; protein aggregate spreading; proteostasis; sequestration; α-synuclein
    DOI:  https://doi.org/10.1016/j.molcel.2025.08.022
  18. Mol Cell. 2025 Sep 03. pii: S1097-2765(25)00708-7. [Epub ahead of print]
    Protection against ferroptosis through maintaining homeostasis of docosahexaenoate-containing phospholipids.
    Yaqin Deng, Goncalo Vale, Yonggang Liang, Shaojie Cui, Shimeng Xu, Jeffrey G McDonald, Jin Ye.
      Although polyunsaturated phospholipids are vital for cellular functions, their overaccumulation renders cells vulnerable to ferroptosis. It remains unclear how cells exposed to excess polyunsaturated fatty acids (PUFAs) prevent their over-incorporation into phospholipids. Here, we identified a mechanism by which ubiquitin regulatory X domain-containing protein 8 (UBXD8), a fatty acid (FA)-interacting protein, prevents overaccumulation of phospholipids containing docosahexaenoate (DHA), one of the most abundant PUFAs in mammalian cells. UBXD8 binds to and activates 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3), which specifically incorporates DHA into phospholipids. Thus, cultured cells and mouse livers deficient in UBXD8 were resistant to ferroptosis because of reduced production of DHA-containing phospholipids. Excess unsaturated FAs, including DHA, through their interaction with UBXD8, disrupt the UBXD8/AGPAT3 complex, thereby inhibiting AGPAT3-catalyzed synthesis of DHA-containing phospholipids. This FA-sensing mechanism prevents overaccumulation of DHA-containing phospholipids in cells exposed to excess DHA, thus reducing the ferroptotic potency of DHA, a property that might contribute to the health benefits of this ω-3 PUFA.
    Keywords:  AA; AGPAT3; DHA; UBXD8; ferroptosis; phospholipids; polyunsaturated fatty acids
    DOI:  https://doi.org/10.1016/j.molcel.2025.08.023
  19. Nat Commun. 2025 Sep 08. 16(1): 7799
    The DNA replication machinery transmits dual signals to prevent unscheduled licensing and execution of centrosome duplication.
    Kyohei Matsuhashi, Kei K Ito, Kaho Nagai, Akira Sanada, Koki Watanabe, Kasuga Takumi, Atsushi Toyoda, Masamitsu Fukuyama, Shohei Yamamoto, Takumi Chinen, Grant S Stewart, Shoji Hata, Daiju Kitagawa.
      Copy number control of DNA and centrosomes is essential for accurate genetic inheritance. DNA replication and centrosome duplication have been recognized as parallel key events for cell division. Here, we discover that the DNA replication machinery directly regulates the licensing and execution processes of centrosome duplication to prevent centrosome amplification. We find that the microcephaly protein DONSON couples DNA replication initiation with Cdc6 translocation to centrosomes. The Cdc6 signal prevents the precocious occurrence of centriole disengagement, the licensing step for centrosome duplication. During DNA replication, DONSON inhibits replisome disassembly by interacting with the CMG helicase, maintaining the intrinsic S/G2 checkpoint signal that blocks centriole-to-centrosome conversion, the execution step for centrosome duplication. Disruption of these dual signals causes precocious centrosome duplication and chromosome mis-segregation, observed in DONSON patient cells. Our results reveal that the DNA replication machinery not only duplicates genetic material but also controls the system for its accurate segregation.
    DOI:  https://doi.org/10.1038/s41467-025-63002-3
  20. Nucleic Acids Res. 2025 Sep 05. pii: gkaf852. [Epub ahead of print]53(17):
    Nuclear compression-mediated DNA damage drives ATR-dependent Lamin expression and mouse ESC differentiation.
    Tanusri Roy, Swetlana Ghosh, Niyati Piplani, Lakshmi Kavitha Sthanam, Niharika Tiwary, Sayak Dhar, W Chingmei Wangsa Konyak, Santosh Surendra Panigrahi, Priya Singh, Divya Tej Sowpati, Sreelaja Nair, Sushil Kumar, P Chandra Shekar, Shamik Sen.
      Embryonic stem cells (ESCs), which are susceptible to DNA damage, depend on a robust and highly efficient DNA damage response (DDR) mechanism for their survival. However, the implications of physical force-mediated DNA damage on ESC fate remain unclear. We show that stiffness-dependent spreading of mouse ESCs (mESCs) induces DNA damage through nuclear compression, with DNA damage causing differentiation through Lamin A/C. Interestingly, differentiation is associated with DNA damage and activation of the DDR factors such as ATR and CHK1. While ATR is typically known to play roles in DDR pathway, its role during stiffness-mediated nuclear compression and mESC differentiation is unknown. While our results show activation of CHK1 pathway and nuclear enrichment of activated ATR on stiff substrates, inhibiting ATR and CHK1 both result in reduction of Lamin A/C expression by different mechanisms. Overall, we demonstrate that mESC differentiation is driven by nuclear compression-mediated DNA damage and involves ATR-dependent modulation of Lamin A/C.
    DOI:  https://doi.org/10.1093/nar/gkaf852
  21. Nature. 2025 Sep 10.
    Energy deficiency selects crowded live epithelial cells for extrusion.
    Saranne J Mitchell, Carlos Pardo-Pastor, Anastassia Tchoumakova, Thomas A Zangle, Jody Rosenblatt.
      Epithelial cells work collectively to provide a protective barrier, yet they turn over rapidly through cell division and death. If the numbers of dividing and dying cells do not match, the barrier can vanish, or tumours can form. Mechanical forces through the stretch-activated ion channel Piezo1 link both of the processes; stretch promotes cell division, whereas crowding triggers live cells to extrude and then die1,2. However, it was not clear what selects a given crowded cell for extrusion. Here we show that the crowded cells with the least energy and membrane potential are selected for extrusion. Crowding triggers sodium (Na+) entry through the epithelial Na+ channel (ENaC), which depolarizes cells. While those with sufficient energy repolarize, those with limited ATP remain depolarized, which, in turn, triggers water egress through the voltage-gated potassium (K+) channels Kv1.1 and Kv1.2 and the chloride (Cl-) channel SWELL1. Transient water loss causes cell shrinkage, amplifying crowding to activate crowding-induced live cell extrusion. Thus, our findings suggest that ENaC acts as a tension sensor that probes for cells with the least energy to extrude and die, possibly damping inadvertent crowding activation of Piezo1 in background cells. We reveal crowding-sensing mechanisms upstream of Piezo1 that highlight water regulation and ion channels as key regulators of epithelial cell turnover.
    DOI:  https://doi.org/10.1038/s41586-025-09514-w
  22. Nat Struct Mol Biol. 2025 Sep 09.
    A snapshot of proteostasis in human oocytes.
    Dimitris Typas.
      
    DOI:  https://doi.org/10.1038/s41594-025-01679-2
  23. Nat Aging. 2025 Sep 10.
    Mitochondria-associated condensates maintain mitochondrial homeostasis and promote lifespan.
    Yan Bai, Tengfei Ma, Shan Zhao, Shalan Li, Xin Wang, Jingyang Li, Wenhao Sun, Yang Yang, Fenglian Liu, Qian Shan, Zizhen Qin, Nan Liu, Jie Zhang, Fei Tian, Mei Duan, Shunkai Chen, Fan Lai, Qingfeng Chen, Xuna Wu, Chonglin Yang.
      Membraneless organelles assembled by liquid-liquid phase separation interact with diverse membranous organelles to regulate distinct cellular processes. It remains unknown how membraneless organelles are engaged in mitochondrial homeostasis. Here we demonstrate that mitochondria-associated translation organelles (MATOs) mediate local synthesis of proteins required for structural and functional maintenance of mitochondria. In Caenorhabditis elegans, the RNA-binding protein LARP-1 (La-related protein 1) orchestrates coalescence of translation machinery and multiple RNA-binding proteins via liquid-liquid phase separation into MATOs that associate with mitochondria in a translocase of the outer membrane complex-dependent manner. LARP-1 deficiency markedly reduces mitochondrial protein levels, impairing cristae organization and ATP production. Specifically, we show that the membrane-shaping MICOS subunit IMMT-1(MIC60) and the ATP synthase β subunit ATP-2, both being important for cristae organization, are synthesized in LARP-1 MATOs. During aging and starvation, LARP-1 MATOs dissociate from mitochondria; however, mitochondrion-persistent LARP-1 MATOs protect mitochondrial health and greatly extend lifespan. These findings suggest an important mitochondrion-regulating mechanism in aging and stress.
    DOI:  https://doi.org/10.1038/s43587-025-00942-x
  24. EMBO Rep. 2025 Sep 08.
    Dynamic association of H3K36me3 with pericentromeric heterochromatin regulates its replication time.
    Sunil Kumar Pradhan, Hui Zhang, Ksenia G Kolobynina, Alexander Rapp, Maria Arroyo, M Cristina Cardoso.
      The flexibility of the spatio-temporal genome replication program during development and disease highlights the regulatory role of plastic epigenetic mechanisms over genetic determinants. Histone post-translational modifications are broadly implicated in replication timing control, yet the specific mechanisms through which individual histone marks influence replication dynamics, particularly in heterochromatin, remain unclear. Here, we demonstrate that H3K36me3 dynamically enriches at pericentromeric heterochromatin, composed of major satellite DNA repeats, prior to replication during mid S phase in mouse embryonic stem cells. By knocking down lysine 36-specific methyltransferases or by targeting the H3K36M oncohistone to pericentromeric heterochromatin, we reduce global or local H3K36me3 levels, respectively, revealing its essential role in preserving the replication timing of constitutive heterochromatin. Loss of H3K36me3 accompanies increased RNA polymerase II serine-5 phosphorylation and lowered major satellite RNA levels, indicating transcriptional dysregulation. Notably, we identify a strand-specific contribution of major satellite forward transcripts in regulating the replication timing of constitutive heterochromatin and maintaining chromatin stability, highlighting the importance of non-coding RNAs as critical regulators of replication timing.
    Keywords:  H3K36me3; MajSat RNA; Oncohistone; Pericentromeric Heterochromatin; Replication Timing
    DOI:  https://doi.org/10.1038/s44319-025-00575-6
  25. Nat Commun. 2025 Sep 12. 16(1): 8267
    Matrix directs trophoblast differentiation in a bioprinted organoid model of early placental development.
    Claire Richards, Hao Chen, Matthew O'Rourke, Ashley Bannister, Grace Owen, Alexander Volkerling, Arnab Ghosh, Catherine A Gorrie, David Gallego-Ortega, Amy L Bottomley, Matthew P Padula, Kristine C McGrath, Louise Cole, Philip M Hansbro, Lana McClements.
      Trophoblast organoids can provide crucial insights into mechanisms of placentation, however their potential is limited by highly variable extracellular matrices unable to reflect in vivo tissues. Here, we present a bioprinted placental organoid model, generated using the first trimester trophoblast cell line, ACH-3P, and a synthetic polyethylene glycol (PEG) matrix. Bioprinted or Matrigel-embedded organoids differentiate spontaneously from cytotrophoblasts into two major subtypes: extravillous trophoblasts (EVTs) and syncytiotrophoblasts (STBs). Bioprinted organoids are driven towards EVT differentiation and show close similarity with early human placenta or primary trophoblast organoids. Inflammation inhibits proliferation and STBs within bioprinted organoids, which aspirin or metformin (0.5 mM) cannot rescue. We reverse the inside-out architecture of ACH-3P organoids by suspension culture with STBs forming on the outer layer of organoids, reflecting placental tissue. Our bioprinted methodology is applicable to trophoblast stem cells. We present a high-throughput, automated, and tuneable trophoblast organoid model that reproducibly mimics the placental microenvironment in health and disease.
    DOI:  https://doi.org/10.1038/s41467-025-62996-0
  26. Cell Rep. 2025 Sep 09. pii: S2211-1247(25)01033-2. [Epub ahead of print]44(9): 116262
    Dynamic regulation of the oxidative stress response by the E3 ligase TRIP12.
    Andrew J Ingersoll, Devlon M McCloud, Jenny Y Hu, Michael Rape.
      Centered on the transcription factor NRF2 and its E3 ligase CUL3KEAP1, the oxidative stress response protects cells from damage by reactive oxygen species (ROS). Increasing ROS inhibits CUL3KEAP1 to stabilize NRF2 and elicit antioxidant gene expression, while cells recovering from stress rapidly turn over NRF2 again to prevent reductive stress and oxeiptosis-dependent death. How cells reinitiate NRF2 degradation after ROS have been cleared remains poorly understood. Here, we identify the essential E3 ligase TRIP12 as a crucial component of the oxidative stress response. TRIP12 is a ubiquitin chain elongation factor that cooperates with CUL3KEAP1 to ensure robust NRF2 degradation. In this manner, TRIP12 accelerates stress response silencing as oxidative stress is being resolved but limits NRF2 activation during stress. The need for dynamic control of NRF2 degradation therefore comes at the cost of diminished stress signaling, suggesting that TRIP12 inhibition could be used to treat degenerative pathologies characterized by ROS accumulation.
    Keywords:  CP: Cancer; CP: Molecular biology; CUL3; KEAP1; NRF2; ROS; TRIP12; oxidative stress response; proteasome; reactive oxygen species; ubiquitin
    DOI:  https://doi.org/10.1016/j.celrep.2025.116262
  27. PLoS Genet. 2025 Sep 08. 21(9): e1011605
    A novel DNA repair-independent role for Gen nuclease in promoting unscheduled polyploidy cell proliferation.
    Manon Budzyk, Anthony Simon, Anne-Sophie Mace, Renata Basto.
      Unscheduled whole genome duplication (WGD), also described as unscheduled or non-physiological polyploidy, can lead to genetic instability and is commonly observed in human cancers. WGD generates DNA damage due to scaling defects between replication factors and DNA content. As a result DNA damage repair mechanisms are thought to be critical for ensuring cell viability and proliferation under these conditions. In this study, we explored the role of homologous recombination and Holliday junction resolution in non-physiological polyploidy in vivo. Using Drosophila genetics and high-resolution imaging, we identified a key and surprising role for Gen/Gen1 nuclease. Our findings revealed that loss-of-function and overexpression of Gen have opposing effects, delaying or accelerating the proliferation of polyploid cells, respectively. These changes ultimately impact cell proliferation, nuclear asynchrony and mitotic DNA damage levels. Surprisingly, our findings show that this effect is unrelated with the expected Gen's function in DNA damage repair. Instead, Gen seems to influence polyploid DNA replication rates. This work identifies a novel function for Gen nuclease and provides new insights into the cellular and molecular requirements of non-physiological polyploidy.
    DOI:  https://doi.org/10.1371/journal.pgen.1011605
  28. Proc Natl Acad Sci U S A. 2025 Sep 16. 122(37): e2510007122
    Structural basis for Rad54- and Hed1-mediated regulation of Rad51 during the transition from mitotic to meiotic recombination.
    Yeonoh Shin, Michael T Petassi, Aidan M Jessop, Stefan Y Kim, Razvan Matei, Katherine Morse, Vivek B Raina, Upasana Roy, Eric C Greene.
      Rad51 catalyzes the DNA pairing reactions that take place during homologous recombination (HR), and HR must be tightly regulated to ensure physiologically appropriate outcomes. Rad54 is an ATP-dependent DNA motor protein that stimulates Rad51 activity during mitosis. In meiosis Rad51 is downregulated by the protein Hed1, which blocks Rad54 binding to Rad51, and allows Dmc1 to function as the active recombinase. We currently have a poor understanding of the regulatory interplay between Rad54, Hed1, Rad51, and Dmc1. Here, we identify a conserved Rad51 interaction motif within Rad54, and we solve a CryoEM structure of this motif bound to Rad51. We also identify a distinct Rad51 interaction motif within Hed1 and solve its structure bound to Rad51. These structures explain how Rad54 engages Rad51 to promote recombination between sister chromatids during mitosis and how Rad51 is downregulated by Hed1 upon entry into meiosis such that its meiosis-specific homolog Dmc1 can promote recombination between homologous chromosomes.
    Keywords:  Hed1; Rad51; Rad54; homologous recombination; meiosis
    DOI:  https://doi.org/10.1073/pnas.2510007122
  29. J Biol Chem. 2025 Sep 08. pii: S0021-9258(25)02547-5. [Epub ahead of print] 110695
    Tubulin hyperacetylation drives HMGB1 nuclear exit via the ROS-PARP1 axis leading to rotenone-induced G2/M Arrest.
    Sourav Dutta, Semanti Chakraborty, Ayushi Ghosh, Priyadarshini Halder, Shubhra Majumder, Ratnadip Paul, Somsubhra Nath, Piyali Mukherjee.
      Rotenone, a lipophilic pesticide, is strongly linked to dopaminergic neuronal loss primarily through mitochondrial complex I inhibition. Beyond its well-characterized neurotoxic effects, rotenone also triggers G2/M arrest in cells, but the molecular mechanisms linking this cell cycle perturbation to neurodegeneration remain unclear. Here, we identify HMGB1 as a key player in this process. HMGB1, known for its roles in genomic integrity and inflammation, exits the nucleus during rotenone-induced G2/M arrest, whereas its nuclear retention protects against mitotic DNA damage and subsequent cell cycle arrest. We found that rotenone-induced tubulin hyperacetylation precedes HMGB1 nuclear exit and is associated with increased mitochondrial ROS (mtROS) levels. Notably, reducing the levels of αTAT1 (alpha tubulin acetyltransferase 1) lowers mtROS production, preventing HMGB1 nuclear exit and subsequent rotenone-induced G2/M arrest. Although ROS is known to enhance tubulin acetylation, our findings reveal a bidirectional relationship in which tubulin acetylation regulates mtROS production and exacerbates cellular oxidative stress. Moreover, the PARP1 inhibitor PJ34 suppresses HMGB1 nuclear exit and rescues G2/M arrest, suggesting that mtROS-induced DNA damage elevates PARP1 activity, driving HMGB1 PARylation and subsequent translocation thus impairing DNA damage repair. Together, our findings uncover a previously unknown tubulin acetylation/mtROS/HMGB1 axis as a key driver of rotenone-induced G2/M arrest, highlighting the essential role of nuclear HMGB1 in maintaining genomic stability. Given that dopaminergic neurons in post-mortem PD brains exhibit G2/M arrest suggestive of abortive cell cycle re-entry, targeting this dysregulated axis may offer a promising strategy to mitigate rotenone-induced neurotoxicity.
    Keywords:  G2/M arrest; Glycirrhizic acid; HMGB1; PJ34; Tubulin acetylation; mitochondrial ROS; rotenone
    DOI:  https://doi.org/10.1016/j.jbc.2025.110695
  30. Cell. 2025 Sep 02. pii: S0092-8674(25)00929-8. [Epub ahead of print]
    HT SpaceM: A high-throughput and reproducible method for small-molecule single-cell metabolomics.
    Jeany Delafiori, Mohammed Shahraz, Andreas Eisenbarth, Volker Hilsenstein, Bernhard Drotleff, Alberto Bailoni, Bishoy Wadie, Måns Ekelöf, Alexander Mattausch, Theodore Alexandrov.
      Single-cell metabolomics (SCM) promises to reveal metabolism in its complexity and heterogeneity, yet current methods struggle with detecting small-molecule metabolites, throughput, and reproducibility. Addressing these gaps, we developed HT SpaceM, a high-throughput SCM method combining cell preparation on custom glass slides, small-molecule matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (MS), and batch processing. We propose a unified framework covering quality control, characterization, structural validation, and differential and functional analyses. Profiling HeLa and NIH3T3 cells, we detected 73 small-molecule metabolites validated by bulk liquid chromatography tandem MS (LC-MS/MS), achieving high reproducibility and single-cell resolution. Interrogating nine NCI-60 cancer cell lines and HeLa, we identified cell-type markers in subpopulations and metabolic hubs. Upon inhibiting glycolysis in HeLa cells, we observed emerging glucose-centered metabolic coordination and intra-condition heterogeneity. Overall, we demonstrate how HT SpaceM enables robust, large-scale SCM across over 140,000 cells from 132 samples and provide guidance on how to interpret metabolic insights beyond population averages.
    Keywords:  LC-MS/MS; MALDI-imaging mass spectrometry; NCI-60; SpaceM; co-abundance; heterogeneity; high-throughput; reproducibility; single-cell metabolomics; small-molecule metabolites
    DOI:  https://doi.org/10.1016/j.cell.2025.08.015
  31. Proc Natl Acad Sci U S A. 2025 Sep 16. 122(37): e2423875122
    Emergent depth-mechanosensing of epithelial collectives regulates cell clustering and dispersal on layered matrices.
    Hongsheng Yu, Amit Pathak.
      During wound healing, tumor growth, and organ formation, epithelial cells migrate and cluster in layered tissue environments. Although cellular mechanosensing of adhered extracellular matrices is now well recognized, it is unclear how deeply cells sense through distant matrix layers. Since single cells can mechanosense stiff basal surfaces through soft hydrogels of <10 μm thickness, here we ask whether cellular collectives can perform such "depth-mechanosensing" through thicker matrix layers. Using a collagen-polyacrylamide double-layer hydrogel, we found that epithelial cell collectives can mechanosense basal substrates at a depth of >100 μm, assessed by cell clustering and collagen deformation. On collagen layers with stiffer basal substrates, cells initially migrate slower while performing higher collagen deformation and stiffening, resulting in reduced dispersal of epithelial clusters. These processes occur in two broad phases: cellular clustering and dynamic collagen deformation, followed by cell migration and dispersal. Using a cell-populated collagen-polyacrylamide computational model, we show that stiffer basal substrates enable higher collagen deformation, which in turn extends the clustering phase of epithelial cells and reduces their dispersal. Disruption of collective collagen deformation, by either α-catenin depletion or myosin-II inhibition, disables the depth-mechanosensitive differences in epithelial responses between soft and stiff basal substrates. These findings suggest that depth-mechanosensing is an emergent property that arises from collective collagen deformation caused by epithelial cell clusters. This work broadens the conventional understanding of epithelial mechanosensing from immediate surfaces to underlying basal matrices, providing insights relevant to tissue contexts with layers of varying stiffness, such as wound healing and tumor invasion.
    Keywords:  collagen; epithelial cells; extracellular matrix; mechanobiology; mechanosensing
    DOI:  https://doi.org/10.1073/pnas.2423875122
  32. Proc Natl Acad Sci U S A. 2025 Sep 16. 122(37): e2517556122
    DNA polymerase α-primase can function as a translesion DNA polymerase.
    Ryan Mayle, Roxana Georgescu, Michael E O'Donnell.
      Replication of cellular chromosomes requires a primase to generate short RNA primers to initiate genomic replication. While bacterial and archaeal primase generate short RNA primers, the eukaryotic primase, Polα-primase, contains both RNA primase and DNA polymerase (Pol) subunits that function together to form a >20 base hybrid RNA-DNA primer. Interestingly, the DNA Pol1 subunit of Polα lacks a 3'-5' proofreading exonuclease, contrary to the high-fidelity normally associated with DNA replication. However, Polδ and Polε synthesize the majority of the eukaryotic genome, and both contain 3'-5' exonuclease activity for high fidelity. Nonetheless, even the small amount of DNA produced by Pol1 in each of the many RNA/DNA primers during chromosome replication adds up to tens of millions of nucleotides in a human genome. Thus, it has been a longstanding question why Pol1 lacks a proofreading exonuclease. We show here that Polα is uniquely capable of traversing common oxidized or hydrolyzed template nucleotides and propose that Polα evolved to bypass these common template lesions when they are encountered during chromosome replication. Additionally, we show a unique ability of replication factor C (RFC) to stimulate Polδ lesion bypass, independent of its sliding clamp. This suggests that there may be a coordination between Polδ and RFC that does not involve RFC loading of PCNA.
    Keywords:  DNA polymerase alpha; DNA repair; DNA replication; DNA translesion bypass; primase
    DOI:  https://doi.org/10.1073/pnas.2517556122
  33. Nat Biotechnol. 2025 Sep 09.
    Genome-wide mapping of RNA-protein associations through sequencing.
    Zhijie Qi, Shuanghong Xue, Junchen Chen, Wenxin Zhao, Kara Johnson, Xingzhao Wen, John Lalith Charles Richard, Pei Lin, Sheng Zhong.
      RNA-protein interactions critically regulate gene expression and cellular processes, yet their comprehensive mapping remains challenging due to their structural diversity. We introduce PRIM-seq (protein-RNA interaction mapping by sequencing), a method for concurrent de novo identification of RNA-binding proteins and their associated RNAs. PRIM-seq generates unique chimeric DNA sequences by proximity ligation of RNAs with protein-linked DNA barcodes, which are subsequently decoded through sequencing. We apply PRIM-seq to two human cell lines and construct a human RNA-protein association network (HuRPA), encompassing >350,000 associations involving ~7,000 RNAs and ~11,000 proteins, including 2,610 proteins that each interact with at least 10 distinct RNAs. We experimentally validate the tumorigenesis-associated lincRNA LINC00339, the RNA with the highest number of protein associations in HuRPA, as a protein-associated RNA. We further validate the RNA-associating abilities of chromatin-conformation regulators SMC1A, SMC3 and RAD21, as well as the metabolic enzyme PHGDH. PRIM-seq enables systematic discovery and prioritization of RNA-binding proteins and their targets without gene- or protein-specific reagents.
    DOI:  https://doi.org/10.1038/s41587-025-02780-z
  34. J Cell Biol. 2025 Oct 06. pii: e202406017. [Epub ahead of print]224(10):
    SEPTIN9 locally activates the RhoGEF ARHGEF18 to promote early stages of mitochondrial fission.
    Rachel Shannon, Yadu Balachandran, Xindi Wang, Maxime Boutry, Hong Xie, Peter K Kim, William S Trimble.
      Mitochondria continually undergo fission to maintain their network and health. Nascent fission sites are marked by the ER, which facilitates actin polymerization to drive calcium flux into the mitochondrion and constrict the inner mitochondrial membrane. Septins are a major eukaryotic cytoskeleton component that forms filaments that can both directly and indirectly modulate other cytoskeleton components, including actin. Septins have been implicated in mitochondrial fission; however, a connection between septins and the regulation of cytoskeletal machinery driving fission is not known. We find that SEPTIN9 is present at mitochondrial fission sites from its early stages with the ER and prior to the fission factor dynamin-related protein 1 (DRP1). SEPTIN9 has an isoform-specific role in fission, dependent on its N-terminal interaction to activate a Rho guanine nucleotide exchange factor, ARHGEF18. Without SEPTIN9, mitochondrial calcium influx is impaired, indicating SEPTIN9-containing octamers play a critical role in the early stages of fission.
    DOI:  https://doi.org/10.1083/jcb.202406017
  35. Nat Biotechnol. 2025 Sep 10.
    A rapid imaging-based screen for induced-proximity degraders identifies a potent degrader of oncoprotein SKP2.
    Yankai Chu, Shishuang Chen, Mingyang Yang, Yin Chen, Huiling Fang, Pinyu Huang, Yueru Xie, Chi Sun, Yun Chen, Baoding Zhang, Li Li, Hongchen Mu, Ding Song, Wentao Cheng, Chao Wang, Wei Jiang, Xiaolan Xu, Zhengjin He, Shuo Chen, Mingxian Liu, Jingchuan Ma, Man Yang, Jiaqi Cao, Jing Gao, Jiali Shen, Lulu Zhang, Yu Bai, Zheyi Liu, Jingyao Chen, Siqi Dai, Yi Arial Zeng, Yun Zhao, Hu Zhou, Chong Chen, Huanwei Ru, Li Tan, Ximin Chi, Fangjun Wang, Daming Gao, Moubin Lin, Xianming Deng, Hai Jiang.
      Targeted protein degraders hold potential as therapeutic agents to target conventionally 'undruggable' proteins. Here, we develop a high-throughput screen, DEath FUSion Escaper (DEFUSE), to identify small-molecule protein degraders. By conjugating the protein of interest to a fast-acting triggerable death protein, this approach translates target protein degradation into a cell survival phenotype to illustrate the presence of degraders. Using this method, we discovered a small molecule (SKPer1) that triggers degradation of the oncoprotein SKP2 and specifically kills SKP2-expressing cancer cells. Mechanistically, SKPer1 acts as an induced-proximity degrader by inducing interaction between SKP2 and an E3 ligase, STUB1, resulting in SKP2 ubiquitination and degradation. SKPer1 exhibits substantial tumour suppression with good safety profiles in vivo. We further show that a sequence of ten amino acids from SKP2 can serve as a versatile degradation tag.
    DOI:  https://doi.org/10.1038/s41587-025-02793-8
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