bims-ginsta Biomed News
on Genome instability
Issue of 2024–07–07
twenty-six papers selected by
Jinrong Hu, National University of Singapore
  1. A transient transcriptional activation governs unpolarized-to-polarized morphogenesis during embryo implantation.
  2. The cell cycle oscillator and spindle length set the speed of chromosome separation in Drosophila embryos.
  3. An activity-specificity trade-off encoded in human transcription factors.
  4. Proximity-based activation of AURORA A by MPS1 potentiates error correction.
  5. DNA break induces rapid transcription repression mediated by proteasome-dependent RNAPII removal.
  6. Forces Shaping the Blastocyst.
  7. Direct observation of translational activation by a ribonucleoprotein granule.
  8. Imaging of Existing and Newly Translated Proteins Elucidates Mechanisms of Sarcomere Turnover.
  9. Neutrophils actively swell to potentiate rapid migration.
  10. Genome-wide quantification of RNA flow across subcellular compartments reveals determinants of the mammalian transcript life cycle.
  11. Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors.
  12. New insights into oocyte cytoplasmic lattice-associated proteins.
  13. PCM1 conveys centrosome asymmetry to polarized endosome dynamics in regulating daughter cell fate.
  14. Maternal mRNA deadenylation is defective in in vitro matured mouse and human oocytes.
  15. DIS3 ribonuclease is essential for spermatogenesis and male fertility in mice.
  16. Premature aging in aneuploid yeast is caused in part by aneuploidy-induced defects in Ribosome Quality Control.
  17. Microtubule forces drive nuclear damage in LMNA cardiomyopathy.
  18. DNA polymerase κ participates in early S-phase DNA replication in human cells.
  19. Mitochondrial Structure and Function in Human Heart Failure.
  20. Next-generation Drosophila protein interactome map and its functional implications.
  21. Semantic encoding during language comprehension at single-cell resolution.
  22. A genome-wide screen links peroxisome regulation with Wnt signaling through RNF146 and TNKS/2.
  23. CCAR1 promotes DNA repair via alternative splicing.
  24. Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids.
  25. The history of chromosomal instability in genome doubled tumors.
  26. Lattice light sheet microscopy reveals 4D force propagation dynamics and leading-edge behaviors in an embryonic epithelium in Drosophila.
  1. Mol Cell. 2024 Jun 27. pii: S1097-2765(24)00483-0. [Epub ahead of print]
    A transient transcriptional activation governs unpolarized-to-polarized morphogenesis during embryo implantation.
    Xuehui Lyu, Yingzi Cui, Yinfei Kong, Min Yang, Hui Shen, Shuyun Liao, Shiyu Li, Chenrui An, Haoyi Wang, Zhe Zhang, Jennie Ong, Yan Li, Peng Du.
      During implantation, embryos undergo an unpolarized-to-polarized transition to initiate postimplantation morphogenesis. However, the underlying molecular mechanism is unknown. Here, we identify a transient transcriptional activation governing embryonic morphogenesis and pluripotency transition during implantation. In naive pluripotent embryonic stem cells (ESCs), which represent preimplantation embryos, we find that the microprocessor component DGCR8 can recognize stem-loop structures within nascent mRNAs to sequester transcriptional coactivator FLII to suppress transcription directly. When mESCs exit from naive pluripotency, the ERK/RSK/P70S6K pathway rapidly activates, leading to FLII phosphorylation and disruption of DGCR8/FLII interaction. Phosphorylated FLII can bind to transcription factor JUN, activating cell migration-related genes to establish poised pluripotency akin to implanting embryos. Resequestration of FLII by DGCR8 drives poised ESCs into formative pluripotency. In summary, we identify a DGCR8/FLII/JUN-mediated transient transcriptional activation mechanism. Disruption of this mechanism inhibits naive-poised-formative pluripotency transition and the corresponding unpolarized-to-polarized transition during embryo implantation, which are conserved in mice and humans.
    Keywords:  DGCR8; ERK; FLII; JUN; embryo implantation; miRNA independent; nascent RNA; pluripotency; stem cells; transcription regulation
    DOI:  https://doi.org/10.1016/j.molcel.2024.06.005
  2. bioRxiv. 2024 Jun 18. pii: 2024.06.17.598879. [Epub ahead of print]
    The cell cycle oscillator and spindle length set the speed of chromosome separation in Drosophila embryos.
    Yitong Xu, Anna Chao, Melissa Rinaldin, Alison Kickuth, Jan Brugués, Stefano Di Talia.
      Anaphase is tightly controlled in space and time to ensure proper separation of chromosomes. The mitotic spindle, the self-organized microtubule structure driving chromosome segregation, scales in size with the available cytoplasm. Yet, the relationship between spindle size and chromosome movement remains poorly understood. Here, we address how the movement of chromosomes changes during the cleavage divisions of the Drosophila blastoderm. We show that the speed of chromosome separation gradually decreases during the 4 nuclear divisions of the blastoderm. This reduction in speed is accompanied by a similar reduction in the length of the spindle, thus ensuring that these two quantities are tightly linked. Using a combination of genetic and quantitative imaging approaches, we find that two processes contribute to controlling the speed at which chromosomes move at mitotic exit: the activity of molecular motors important for microtubule depolymerization and the cell cycle oscillator. Specifically, we found that the levels of Klp10A, Klp67A, and Klp59C, three kinesin-like proteins important for microtubule depolymerization, contribute to setting the speed of chromosome separation. This observation is supported by quantification of microtubule dynamics indicating that poleward flux rate scales with the length of the spindle. Perturbations of the cell cycle oscillator using heterozygous mutants of mitotic kinases and phosphatases revealed that the duration of anaphase increases during the blastoderm cycles and is the major regulator of chromosome velocity. Thus, our work suggests a potential link between the biochemical rate of mitotic exit and the forces exerted by the spindle. Collectively, we propose that the cell cycle oscillator and spindle length set the speed of chromosome separation in anaphase.
    DOI:  https://doi.org/10.1101/2024.06.17.598879
  3. Nat Cell Biol. 2024 Jul 05.
    An activity-specificity trade-off encoded in human transcription factors.
    Julian Naderi, Alexandre P Magalhaes, Gözde Kibar, Gregoire Stik, Yaotian Zhang, Sebastian D Mackowiak, Hannah M Wieler, Francesca Rossi, Rene Buschow, Marie Christou-Kent, Marc Alcoverro-Bertran, Thomas Graf, Martin Vingron, Denes Hnisz.
      Transcription factors (TFs) control specificity and activity of gene transcription, but whether a relationship between these two features exists is unclear. Here we provide evidence for an evolutionary trade-off between the activity and specificity in human TFs encoded as submaximal dispersion of aromatic residues in their intrinsically disordered protein regions. We identified approximately 500 human TFs that encode short periodic blocks of aromatic residues in their intrinsically disordered regions, resembling imperfect prion-like sequences. Mutation of periodic aromatic residues reduced transcriptional activity, whereas increasing the aromatic dispersion of multiple human TFs enhanced transcriptional activity and reprogramming efficiency, promoted liquid-liquid phase separation in vitro and more promiscuous DNA binding in cells. Together with recent work on enhancer elements, these results suggest an important evolutionary role of suboptimal features in transcriptional control. We propose that rational engineering of amino acid features that alter phase separation may be a strategy to optimize TF-dependent processes, including cellular reprogramming.
    DOI:  https://doi.org/10.1038/s41556-024-01411-0
  4. bioRxiv. 2024 Jun 13. pii: 2024.06.11.598300. [Epub ahead of print]
    Proximity-based activation of AURORA A by MPS1 potentiates error correction.
    Nelson Leça, Francisca Barbosa, Sergi Rodriguez-Calado, Margarida Moura, Paulo D Pedroso, Inês Pinto, Arianna E Verza, Tanja Bange, Claudio E Sunkel, Marin Barisic, Thomas J Maresca, Carlos Conde.
      Faithfull cell division relies on mitotic chromosomes becoming bioriented with each pair of sister kinetochores bound to microtubules oriented toward opposing spindle poles. Erroneous kinetochore-microtubule attachments often form during early mitosis, but are destabilized through the phosphorylation of outer kinetochore proteins by centromeric AURORA B kinase (ABK) and centrosomal AURORA A kinase (AAK), thus allowing for re-establishment of attachments until biorientation is achieved. MPS1-mediated phosphorylation of NDC80 has also been shown to directly weaken the kinetochore-microtubule interface in yeast. In human cells, MPS1 has been proposed to transiently accumulate at end-on attached kinetochores and phosphorylate SKA3 to promote microtubule release. Whether MPS1 directly targets NDC80 and/or promotes the activity of AURORA kinases in metazoans remains unclear. Here, we report a novel mechanism involving communication between kinetochores and centrosomes, wherein MPS1 acts upstream of AAK to promote error correction. MPS1 on pole-proximal kinetochores phosphorylates the C-lobe of AAK thereby increasing its activation at centrosomes. This proximity-based activation ensures the establishment of a robust AAK activity gradient that locally destabilizes mal-oriented kinetochores near spindle poles. Accordingly, MPS1 depletion from Drosophila cells causes severe chromosome misalignment and erroneous kinetochore-microtubule attachments, which can be rescued by tethering either MPS1 or constitutively active AAK mutants to centrosomes. Proximity-based activation of AAK by MPS1 also occurs in human cells to promote AAK-mediated phosphorylation of the NDC80 N-terminal tail. These findings uncover an MPS1-AAK cross-talk that is required for efficient error correction, showcasing the ability of kinetochores to modulate centrosome outputs to ensure proper chromosome segregation.
    DOI:  https://doi.org/10.1101/2024.06.11.598300
  5. Cell Rep. 2024 Jul 01. pii: S2211-1247(24)00749-6. [Epub ahead of print]43(7): 114420
    DNA break induces rapid transcription repression mediated by proteasome-dependent RNAPII removal.
    Shuaixin He, Zhiyuan Huang, Yang Liu, Taekjip Ha, Bin Wu.
      A DNA double-strand break (DSB) jeopardizes genome integrity and endangers cell viability. Actively transcribed genes are particularly detrimental if broken and need to be repressed. However, it remains elusive how fast the repression is initiated and how far it influences the neighboring genes on the chromosome. We adopt a recently developed, very fast CRISPR to generate a DSB at a specific genomic locus with precise timing, visualize transcription in live cells, and measure the RNA polymerase II (RNAPII) occupancy near the broken site. We observe that a single DSB represses the transcription of the damaged gene in minutes, which coincides with the recruitment of a damage repair protein. Transcription repression propagates bi-directionally along the chromosome from the DSB for hundreds of kilobases, and proteasome is evoked to remove RNAPII in this process. Our method builds a foundation to measure the rapid kinetic events around a single DSB and elucidate the molecular mechanism.
    Keywords:  CP: Molecular biology; CRISPR-Cas9; DNA damage repair; proteasome; single molecule; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2024.114420
  6. Cold Spring Harb Perspect Biol. 2024 Jul 01. pii: a041519. [Epub ahead of print]
    Forces Shaping the Blastocyst.
    David Rozema, Jean-Léon Maître.
      The blastocyst forms during the first days of mammalian development. The structure of the blastocyst is conserved among placental mammals and is paramount to the establishment of the first mammalian lineages. The blastocyst is composed of an extraembryonic epithelium, the trophectoderm (TE), that envelopes a fluid-filled lumen and the inner cell mass (ICM). To shape the blastocyst, embryos transit through three stages driven by forces that have been characterized in the mouse embryo over the past decade. The morphogenetically quiescent cleavage stages mask dynamic cytoskeletal remodeling. Then, during the formation of the morula, cells pull themselves together and the strongest ones internalize. Finally, the blastocyst forms after the pressurized lumen breaks the radial symmetry of the embryo before expanding in cycles of collapses and regrowth. In this review, we delineate the force patterns sculpting the blastocyst, based on our knowledge on the mouse and, to some extent, human embryos.
    DOI:  https://doi.org/10.1101/cshperspect.a041519
  7. Nat Cell Biol. 2024 Jul 04.
    Direct observation of translational activation by a ribonucleoprotein granule.
    Ruoyu Chen, William Stainier, Jeremy Dufourt, Mounia Lagha, Ruth Lehmann.
      Biomolecular condensates organize biochemical processes at the subcellular level and can provide spatiotemporal regulation within a cell. Among these, ribonucleoprotein (RNP) granules are storage hubs for translationally repressed mRNA. Whether RNP granules can also activate translation and how this could be achieved remains unclear. Here, using single-molecule imaging, we demonstrate that the germ cell-determining RNP granules in Drosophila embryos are sites for active translation of nanos mRNA. Nanos translation occurs preferentially at the germ granule surface with the 3' UTR buried within the granule. Smaug, a cytosolic RNA-binding protein, represses nanos translation, which is relieved when Smaug is sequestered to the germ granule by the scaffold protein Oskar. Together, our findings uncover a molecular process by which RNP granules achieve localized protein synthesis through the compartmentalized loss of translational repression.
    DOI:  https://doi.org/10.1038/s41556-024-01452-5
  8. Circ Res. 2024 Jul 04.
    Imaging of Existing and Newly Translated Proteins Elucidates Mechanisms of Sarcomere Turnover.
    Guy Douvdevany, Itai Erlich, Lilac Haimovich-Caspi, Tomer Mashiah, Maksymilian Prondzynski, Maria Rosaria Pricolo, Jorge Alegre-Cebollada, Wolfgang A Linke, Lucie Carrier, Izhak Kehat.
       BACKGROUND: How the sarcomeric complex is continuously turned over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres.
    METHODS: We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands.
    RESULTS: We disprove the prevailing protein pool model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age and that proteolytic extraction is a rate-limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells.
    CONCLUSIONS: Our findings establish a unidirectional replacement model for cardiac sarcomeres subunit replacement and identify their turnover principles.
    Keywords:  cells, cultured; connectin; muscle proteins; myocytes, cardiac; sarcomeres
    DOI:  https://doi.org/10.1161/CIRCRESAHA.123.323819
  9. Elife. 2024 Jul 02. pii: RP90551. [Epub ahead of print]12
    Neutrophils actively swell to potentiate rapid migration.
    Tamas L Nagy, Evelyn Strickland, Orion D Weiner.
      While the involvement of actin polymerization in cell migration is well-established, much less is known about the role of transmembrane water flow in cell motility. Here, we investigate the role of water influx in a prototypical migrating cell, the neutrophil, which undergoes rapid, directed movement to sites of injury, and infection. Chemoattractant exposure both increases cell volume and potentiates migration, but the causal link between these processes are not known. We combine single-cell volume measurements and a genome-wide CRISPR screen to identify the regulators of chemoattractant-induced neutrophil swelling, including NHE1, AE2, PI3K-gamma, and CA2. Through NHE1 inhibition in primary human neutrophils, we show that cell swelling is both necessary and sufficient for the potentiation of migration following chemoattractant stimulation. Our data demonstrate that chemoattractant-driven cell swelling complements cytoskeletal rearrangements to enhance migration speed.
    Keywords:  cell biology; cell migration; cell size; cell volume; human; neutrophil; physical forces; physics of living systems
    DOI:  https://doi.org/10.7554/eLife.90551
  10. Mol Cell. 2024 Jun 27. pii: S1097-2765(24)00511-2. [Epub ahead of print]
    Genome-wide quantification of RNA flow across subcellular compartments reveals determinants of the mammalian transcript life cycle.
    Robert Ietswaart, Brendan M Smalec, Albert Xu, Karine Choquet, Erik McShane, Ziad Mohamoud Jowhar, Chantal K Guegler, Autum R Baxter-Koenigs, Emma R West, Becky Xu Hua Fu, Luke Gilbert, Stephen N Floor, L Stirling Churchman.
      Dissecting the regulatory mechanisms controlling mammalian transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm in human and mouse cells. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA-binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered a link between DDX3X and nuclear export. For hundreds of RNA metabolism genes, most transcripts with retained introns were degraded by the nuclear exosome, while the remaining molecules were exported with stable cytoplasmic lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, machine learning identified molecular features that predicted the diverse life cycles of mRNAs.
    Keywords:  DDX3X; RNA dynamics; RNA flow; RNA-binding proteins; kinetic modeling; nuclear RNA degradation; nuclear export; poly(A) tails; subcellular TimeLapse-seq; translation
    DOI:  https://doi.org/10.1016/j.molcel.2024.06.008
  11. Nat Commun. 2024 Jul 04. 15(1): 5611
    Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors.
    Qing Hu, Jose Espejo Valle-Inclán, Rashmi Dahiya, Alison Guyer, Alice Mazzagatti, Elizabeth G Maurais, Justin L Engel, Huiming Lu, Anthony J Davis, Isidro Cortés-Ciriano, Peter Ly.
      Mitotic errors generate micronuclei entrapping mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates diverse genomic rearrangements associated with human diseases. How specific repair pathways recognize and process these lesions remains poorly understood. Here we use CRISPR/Cas9 to systematically inactivate distinct DSB repair pathways and interrogate the rearrangement landscape of fragmented chromosomes. Deletion of canonical non-homologous end joining (NHEJ) components substantially reduces complex rearrangements and shifts the rearrangement landscape toward simple alterations without the characteristic patterns of chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within sub-nuclear micronuclei bodies (MN bodies) and undergo ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments are rarely engaged by alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics, persistent 53BP1-labeled MN bodies, and cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements from mitotic errors.
    DOI:  https://doi.org/10.1038/s41467-024-49985-5
  12. Trends Genet. 2024 Jul 01. pii: S0168-9525(24)00149-5. [Epub ahead of print]
    New insights into oocyte cytoplasmic lattice-associated proteins.
    Carlo Giaccari, Francesco Cecere, Lucia Argenziano, Angela Pagano, Andrea Riccio.
      Oocyte maturation and preimplantation embryo development are critical to successful pregnancy outcomes and the correct establishment and maintenance of genomic imprinting. Thanks to novel technologies and omics studies in human patients and mouse models, the importance of the proteins associated with the cytoplasmic lattices (CPLs), highly abundant structures found in the cytoplasm of mammalian oocytes and preimplantation embryos, in the maternal to zygotic transition is becoming increasingly evident. This review highlights the recent discoveries on the role of these proteins in protein storage and other oocyte cytoplasmic processes, epigenetic reprogramming, and zygotic genome activation (ZGA). A better comprehension of these events may significantly improve clinical diagnosis and pave the way for targeted interventions aiming to correct or mitigate female fertility issues and genomic imprinting disorders.
    Keywords:  UHRF1; cytoplasmic lattices; genomic imprinting disorders; oocyte maturation; subcortical maternal complex; zygotic genome activation
    DOI:  https://doi.org/10.1016/j.tig.2024.06.002
  13. bioRxiv. 2024 Jun 18. pii: 2024.06.17.599416. [Epub ahead of print]
    PCM1 conveys centrosome asymmetry to polarized endosome dynamics in regulating daughter cell fate.
    Xiang Zhao, Yiqi Wang, Vincent Mouilleau, Ahmet Can Solak, Jason Garcia, Xingye Chen, Christopher J Wilkinson, Loic Royer, Zhiqiang Dong, Su Guo.
      Vertebrate radial glia progenitors (RGPs), the principal neural stem cells, balance self-renewal and differentiation through asymmetric cell division (ACD), during which unequal inheritance of centrosomes is observed. Mechanistically, how centrosome asymmetry leads to distinct daughter cell fate remains largely unknown. Here we find that the centrosome protein Pericentriolar Material 1 (Pcm1), asymmetrically distributed at the centrosomes, regulates polarized endosome dynamics and RGP fate. In vivo time-lapse imaging and nanoscale-resolution expansion microscopy of zebrafish embryonic RGPs detect Pcm1 on Notch ligand-containing endosomes, in a complex with the polarity regulator Par-3 and dynein motor. Loss of pcm1 disrupts endosome dynamics, with clonal analysis uncovering increased neuronal production at the expense of progenitors. Pcm1 facilitates an exchange of Rab5b (early) for Rab11a (recycling) endosome markers and promotes the formation of Par-3 and dynein macromolecular complexes on recycling endosomes. Finally, in human-induced pluripotent stem cell-derived brain organoids, PCM1 shows asymmetry and co-localization with PARD3 and RAB11A in mitotic neural progenitors. Our data reveal a new mechanism by which centrosome asymmetry is conveyed by Pcm1 to polarize endosome dynamics and Notch signaling in regulating ACD and progenitor fate.
    DOI:  https://doi.org/10.1101/2024.06.17.599416
  14. Nat Commun. 2024 Jul 02. 15(1): 5550
    Maternal mRNA deadenylation is defective in in vitro matured mouse and human oocytes.
    Yusheng Liu, Wenrong Tao, Shuang Wu, Yiwei Zhang, Hu Nie, Zhenzhen Hou, Jingye Zhang, Zhen Yang, Zi-Jiang Chen, Jiaqiang Wang, Falong Lu, Keliang Wu.
      Oocyte in vitro maturation is a technique in assisted reproductive technology. Thousands of genes show abnormally high expression in in vitro maturated metaphase II (MII) oocytes compared to those matured in vivo in bovines, mice, and humans. The mechanisms underlying this phenomenon are poorly understood. Here, we use poly(A) inclusive RNA isoform sequencing (PAIso-seq) for profiling the transcriptome-wide poly(A) tails in both in vivo and in vitro matured mouse and human oocytes. Our results demonstrate that the observed increase in maternal mRNA abundance is caused by impaired deadenylation in in vitro MII oocytes. Moreover, the cytoplasmic polyadenylation of dormant Btg4 and Cnot7 mRNAs, which encode key components of deadenylation machinery, is impaired in in vitro MII oocytes, contributing to reduced translation of these deadenylase machinery components and subsequently impaired global maternal mRNA deadenylation. Our findings highlight impaired maternal mRNA deadenylation as a distinct molecular defect in in vitro MII oocytes.
    DOI:  https://doi.org/10.1038/s41467-024-49695-y
  15. Development. 2024 Jul 01. pii: dev202579. [Epub ahead of print]151(13):
    DIS3 ribonuclease is essential for spermatogenesis and male fertility in mice.
    Zhengpin Wang, Di Wu, Xiaojiang Xu, Guoyun Yu, Nana Li, Xiao Wang, Jian-Liang Li, Jurrien Dean.
      Spermatogonial stem cell (SSC) self-renewal and differentiation provide foundational support for long-term, steady-state spermatogenesis in mammals. Here, we have investigated the essential role of RNA exosome associated DIS3 ribonuclease in maintaining spermatogonial homeostasis and facilitating germ cell differentiation. We have established male germ-cell Dis3 conditional knockout (cKO) mice in which the first and subsequent waves of spermatogenesis are disrupted. This leads to a Sertoli cell-only phenotype and sterility in adult male mice. Bulk RNA-seq documents that Dis3 deficiency partially abolishes RNA degradation and causes significant increases in the abundance of transcripts. This also includes pervasively transcribed PROMoter uPstream Transcripts (PROMPTs), which accumulate robustly in Dis3 cKO testes. In addition, scRNA-seq analysis indicates that Dis3 deficiency in spermatogonia significantly disrupts RNA metabolism and gene expression, and impairs early germline cell development. Overall, we document that exosome-associated DIS3 ribonuclease plays crucial roles in maintaining early male germ cell lineage in mice.
    Keywords:  DIS3; Male fertility; Spermatogenesis
    DOI:  https://doi.org/10.1242/dev.202579
  16. bioRxiv. 2024 Jun 23. pii: 2024.06.22.600216. [Epub ahead of print]
    Premature aging in aneuploid yeast is caused in part by aneuploidy-induced defects in Ribosome Quality Control.
    Leah E Escalante, James Hose, Hollis Howe, Norah Paulsen, Michael Place, Audrey P Gasch.
      Premature aging is a hallmark of Down syndrome, caused by trisomy of human chromosome 21, but the reason is unclear and difficult to study in humans. We used an aneuploid model in wild yeast to show that chromosome amplification disrupts nutrient-induced cell-cycle arrest, quiescence entry, and healthy aging, across genetic backgrounds and amplified chromosomes. We discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). Compared to euploids, aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates. Although they have normal proteasome capacity, aneuploids show signs of ubiquitin dysregulation, which impacts cyclin abundance to disrupt arrest. Remarkably, inducing ribosome stalling in euploids produces similar aberrations, while up-regulating limiting RQC subunits or proteins in ubiquitin metabolism alleviates many of the aneuploid defects. Our results provide implications for other aneuploidy disorders including Down syndrome.
    DOI:  https://doi.org/10.1101/2024.06.22.600216
  17. bioRxiv. 2024 Jun 21. pii: 2024.02.10.579774. [Epub ahead of print]
    Microtubule forces drive nuclear damage in LMNA cardiomyopathy.
    Daria Amiad Pavlov, Carmen Suay Corredera, Mohammad Dehghany, Julie Heffler, Kaitlyn M Shen, Noam Zuela-Sopilniak, Rani Randell, Keita Uchida, Rajan Jain, Vivek Shenoy, Jan Lammerding, Benjamin Prosser.
      Nuclear homeostasis requires a balance of forces between the cytoskeleton and nucleus. Variants in LMNA disrupt this balance by weakening the nuclear lamina, resulting in nuclear damage in contractile tissues and ultimately muscle disease. Intriguingly, disrupting the LINC complex that connects the cytoskeleton to the nucleus has emerged as a promising strategy to ameliorate LMNA cardiomyopathy. Yet how LINC disruption protects the cardiomyocyte nucleus remains unclear. To address this, we developed an assay to quantify the coupling of cardiomyocyte contraction to nuclear deformation and interrogated its dependence on the lamina and LINC complex. We found that the LINC complex was surprisingly dispensable for transferring the majority of contractile strain into the nucleus, and that increased nuclear strain in Lmna- deficient myocytes was not rescued by LINC disruption. However, LINC disruption eliminated the cage of microtubules encircling the nucleus, and disrupting microtubules was sufficient to prevent nuclear damage induced by LMNA deficiency. Through computational modeling we simulated the mechanical stress fields surrounding cardiomyocyte nuclei and show how microtubule compression exploits local vulnerabilities to damage LMNA -deficient nuclei. Our work pinpoints localized, microtubule-dependent force transmission through the LINC complex as a pathological driver and therapeutic target for LMNA cardiomyopathy.
    Graphical abstract:
    DOI:  https://doi.org/10.1101/2024.02.10.579774
  18. Proc Natl Acad Sci U S A. 2024 Jul 09. 121(28): e2405473121
    DNA polymerase κ participates in early S-phase DNA replication in human cells.
    Feng Tang, Yinan Wang, Ting Zhao, Jun Yuan, Andrew H Kellum, Yinsheng Wang.
      Cycling cells replicate their DNA during the S phase through a defined temporal program known as replication timing. Mutation frequencies, epigenetic chromatin states, and transcriptional activities are different for genomic regions that are replicated early and late in the S phase. Here, we found from ChIP-Seq analysis that DNA polymerase (Pol) κ is enriched in early-replicating genomic regions in HEK293T cells. In addition, by feeding cells with N 2-heptynyl-2'-deoxyguanosine followed by click chemistry-based enrichment and high-throughput sequencing, we observed elevated Pol κ activities in genomic regions that are replicated early in the S phase. On the basis of the established functions of Pol κ in accurate and efficient nucleotide insertion opposite endogenously induced N 2-modified dG lesions, our work suggests that active engagement of Pol κ may contribute to diminished mutation rates observed in early-replicating regions of the human genome, including cancer genomes. Together, our work expands the functions of Pol κ and offered a plausible mechanism underlying replication timing-dependent mutation accrual in the human genome.
    Keywords:  DNA replication; mutagenesis; replication timing; translesion synthesis polymerase
    DOI:  https://doi.org/10.1073/pnas.2405473121
  19. Circ Res. 2024 Jul 05. 135(2): 372-396
    Mitochondrial Structure and Function in Human Heart Failure.
    Antentor Hinton, Steven M Claypool, Kit Neikirk, Nanami Senoo, Celestine N Wanjalla, Annet Kirabo, Clintoria R Williams.
      Despite clinical and scientific advancements, heart failure is the major cause of morbidity and mortality worldwide. Both mitochondrial dysfunction and inflammation contribute to the development and progression of heart failure. Although inflammation is crucial to reparative healing following acute cardiomyocyte injury, chronic inflammation damages the heart, impairs function, and decreases cardiac output. Mitochondria, which comprise one third of cardiomyocyte volume, may prove a potential therapeutic target for heart failure. Known primarily for energy production, mitochondria are also involved in other processes including calcium homeostasis and the regulation of cellular apoptosis. Mitochondrial function is closely related to morphology, which alters through mitochondrial dynamics, thus ensuring that the energy needs of the cell are met. However, in heart failure, changes in substrate use lead to mitochondrial dysfunction and impaired myocyte function. This review discusses mitochondrial and cristae dynamics, including the role of the mitochondria contact site and cristae organizing system complex in mitochondrial ultrastructure changes. Additionally, this review covers the role of mitochondria-endoplasmic reticulum contact sites, mitochondrial communication via nanotunnels, and altered metabolite production during heart failure. We highlight these often-neglected factors and promising clinical mitochondrial targets for heart failure.
    Keywords:  cardiovascular diseases; heart failure; hypertension; mitochondria; myocardium
    DOI:  https://doi.org/10.1161/CIRCRESAHA.124.323800
  20. Dev Cell. 2024 Jun 27. pii: S1534-5807(24)00380-0. [Epub ahead of print]
    Next-generation Drosophila protein interactome map and its functional implications.
    Guruharsha Bhat, Kejie Li, George Locke, Marina Theodorou, Krishna Kilambi, Kazuya Hori, Diana Ho, Robert Obar, Leah Williams, Hannah Parzen, Noah Dephoure, Craig Braun, Marc Muskavitch, Susan E Celniker, Steven Gygi, Spyros Artavanis-Tsakonas.
      We describe a next-generation Drosophila protein interaction map-"DPIM2"-established from affinity purification-mass spectrometry of 5,805 baits, covering the largest fraction of the Drosophila proteome. The network contains 32,668 interactions among 3,644 proteins, organized into 632 clusters representing putative functional modules. Our analysis expands the pool of known protein interactions in Drosophila, provides annotation for poorly studied genes, and postulates previously undescribed protein interaction relationships. The predictive power and functional relevance of this network are probed through the lens of the Notch signaling pathway, and we find that newly identified members of complexes that include known Notch modifiers can also modulate Notch signaling. DPIM2 allows direct comparisons with a recently published human protein interaction network, defining the existence of functional interactions conserved across species. Thus, DPIM2 defines a valuable resource for predicting protein co-complex memberships and functional associations as well as generates functional hypotheses regarding specific protein interactions.
    Keywords:  affinity purification; interactome; interologs; modifiers; notch pathway; protein interaction; proteome
    DOI:  https://doi.org/10.1016/j.devcel.2024.06.002
  21. Nature. 2024 Jul 03.
    Semantic encoding during language comprehension at single-cell resolution.
    Mohsen Jamali, Benjamin Grannan, Jing Cai, Arjun R Khanna, William Muñoz, Irene Caprara, Angelique C Paulk, Sydney S Cash, Evelina Fedorenko, Ziv M Williams.
      From sequences of speech sounds1,2 or letters3, humans can extract rich and nuanced meaning through language. This capacity is essential for human communication. Yet, despite a growing understanding of the brain areas that support linguistic and semantic processing4-12, the derivation of linguistic meaning in neural tissue at the cellular level and over the timescale of action potentials remains largely unknown. Here we recorded from single cells in the left language-dominant prefrontal cortex as participants listened to semantically diverse sentences and naturalistic stories. By tracking their activities during natural speech processing, we discover a fine-scale cortical representation of semantic information by individual neurons. These neurons responded selectively to specific word meanings and reliably distinguished words from nonwords. Moreover, rather than responding to the words as fixed memory representations, their activities were highly dynamic, reflecting the words' meanings based on their specific sentence contexts and independent of their phonetic form. Collectively, we show how these cell ensembles accurately predicted the broad semantic categories of the words as they were heard in real time during speech and how they tracked the sentences in which they appeared. We also show how they encoded the hierarchical structure of these meaning representations and how these representations mapped onto the cell population. Together, these findings reveal a finely detailed cortical organization of semantic representations at the neuron scale in humans and begin to illuminate the cellular-level processing of meaning during language comprehension.
    DOI:  https://doi.org/10.1038/s41586-024-07643-2
  22. J Cell Biol. 2024 Oct 07. pii: e202312069. [Epub ahead of print]223(10):
    A genome-wide screen links peroxisome regulation with Wnt signaling through RNF146 and TNKS/2.
    Jonathan T Vu, Katherine U Tavasoli, Connor J Sheedy, Soham P Chowdhury, Lori Mandjikian, Julien Bacal, Meghan A Morrissey, Chris D Richardson, Brooke M Gardner.
      Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerases TNKS and TNKS2, which bind the peroxisomal membrane protein PEX14. We propose that RNF146 and TNKS/2 regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased TNKS/2 and RNF146-dependent degradation of non-peroxisomal substrates, including the β-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of β-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation but also a novel role in bridging peroxisome function with Wnt/β-catenin signaling during development.
    DOI:  https://doi.org/10.1083/jcb.202312069
  23. Mol Cell. 2024 Jul 02. pii: S1097-2765(24)00514-8. [Epub ahead of print]
    CCAR1 promotes DNA repair via alternative splicing.
    Mehmet E Karasu, Leonard Jahnke, Brian J Joseph, Yerkezhan Amerzhanova, Aleksei Mironov, Xuan Shu, Markus S Schröder, Ana Gvozdenovic, Irene Sala, Mihaela Zavolan, Stefanie Jonas, Jacob E Corn.
      DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.
    Keywords:  CRISPR screen; DNA damage response; DNA repair; Fanconi anemia; alternative splicing; mRNA splicing; poison exon; spliceosome
    DOI:  https://doi.org/10.1016/j.molcel.2024.06.011
  24. Nat Mater. 2024 Jul 04.
    Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids.
    Bauer L LeSavage, Daiyao Zhang, Carla Huerta-López, Aidan E Gilchrist, Brad A Krajina, Kasper Karlsson, Amber R Smith, Kremena Karagyozova, Katarina C Klett, Michelle S Huang, Christopher Long, Gernot Kaber, Christopher M Madl, Paul L Bollyky, Christina Curtis, Calvin J Kuo, Sarah C Heilshorn.
      Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix. However, how the altered cell/extracellular-matrix signalling contributes to the PDAC tumour phenotype has been difficult to dissect. Here we design and engineer matrices that recapitulate the key hallmarks of the PDAC tumour extracellular matrix to address this knowledge gap. We show that patient-derived PDAC organoids from three patients develop resistance to several clinically relevant chemotherapies when cultured within high-stiffness matrices mechanically matched to in vivo tumours. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, matrix-induced chemoresistance occurs within a stiff environment due to the increased expression of drug efflux transporters mediated by CD44 receptor interactions with hyaluronan. Moreover, PDAC chemoresistance is reversible following transfer from high- to low-stiffness matrices, suggesting that targeting the fibrotic extracellular matrix may sensitize chemoresistant tumours. Overall, our findings support the potential of engineered matrices and patient-derived organoids for elucidating extracellular matrix contributions to human disease pathophysiology.
    DOI:  https://doi.org/10.1038/s41563-024-01908-x
  25. Cancer Discov. 2024 Jul 01.
    The history of chromosomal instability in genome doubled tumors.
    Toby M Baker, Siqi Lai, Andrew R Lynch, Tom Lesluyes, Haixi Yan, Huw A Ogilvie, Annelien Verfaillie, Stefan Dentro, Amy L Bowes, Nischalan Pillay, Adrienne M Flanagan, Charles Swanton, Paul T Spellman, Maxime Tarabichi, Peter Van Loo.
      Tumors frequently display high chromosomal instability and contain multiple copies of genomic regions. Here, we describe GRITIC, a generic method for timing genomic gains leading to complex copy number states, using single-sample bulk whole-genome sequencing data. By applying GRITIC to 6,091 tumors, we found that non-parsimonious evolution is frequent in the formation of complex copy number states in genome-doubled tumors. We measured chromosomal instability before and after genome duplication in human tumors and found that late genome doubling was followed by an increase in the rate of copy number gain. Copy number gains often accumulate as punctuated bursts, commonly after genome doubling. We infer that genome duplications typically affect the landscape of copy number losses, while only minimally impacting copy number gains. In summary, GRITIC is a novel copy number gain timing framework that permits the analysis of copy number evolution in chromosomally unstable tumors.
    DOI:  https://doi.org/10.1158/2159-8290.CD-23-1249
  26. Curr Biol. 2024 Jun 27. pii: S0960-9822(24)00777-2. [Epub ahead of print]
    Lattice light sheet microscopy reveals 4D force propagation dynamics and leading-edge behaviors in an embryonic epithelium in Drosophila.
    Timothy E Vanderleest, Yi Xie, Rashmi Budhathoki, Katie Linvill, Chad Hobson, John Heddleston, Dinah Loerke, J Todd Blankenship.
      How pulsed contractile dynamics drive the remodeling of cell and tissue topologies in epithelial sheets has been a key question in development and disease. Due to constraints in imaging and analysis technologies, studies that have described the in vivo mechanisms underlying changes in cell and neighbor relationships have largely been confined to analyses of planar apical regions. Thus, how the volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface in three dimensions, including especially the apical-basal axis, is unclear. Here, we perform lattice light sheet microscopy (LLSM)-based analysis to determine how far and fast forces propagate across different apical-basal layers, as well as where topological changes initiate from in a columnar epithelium. These datasets are highly time- and depth-resolved and reveal that topology-changing forces are spatially entangled, with contractile force generation occurring across the observed apical-basal axis in a pulsed fashion, while the conservation of cell volumes constrains instantaneous cell deformations. Leading layer behaviors occur opportunistically in response to favorable phasic conditions, with lagging layers "zippering" to catch up as new contractile pulses propel further changes in cell topologies. These results argue against specific zones of topological initiation and demonstrate the importance of systematic 4D-based analysis in understanding how forces and deformations in cell dimensions propagate in a three-dimensional environment.
    Keywords:  Epithelium; convergent extension; force propagation; lattice light sheet; morphogenesis; oscillations; tissue extension
    DOI:  https://doi.org/10.1016/j.cub.2024.06.017
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