bims-ginsta Biomed News
on Genome instability
Issue of 2024–08–11
29 papers selected by
Jinrong Hu, National University of Singapore
  1. Hox gene activity directs physical forces to differentially shape chick small and large intestinal epithelia.
  2. Diversity of ribosomes at the level of rRNA variation associated with human health and disease.
  3. Redox heterogeneity in mouse embryonic stem cells individualizes cell fate decisions.
  4. Stable and robust Xi and Y transcriptomes drive cell-type-specific autosomal and Xa responses in vivo and in vitro in four human cell types.
  5. Age-associated clonal B cells drive B cell lymphoma in mice.
  6. p16-dependent increase of PD-L1 stability regulates immunosurveillance of senescent cells.
  7. A spatial expression atlas of the adult human proximal small intestine.
  8. Active shape programming drives Drosophila wing disc eversion.
  9. Membrane prewetting by condensates promotes tight-junction belt formation.
  10. Cyclin B3 is a dominant fast-acting cyclin that drives rapid early embryonic mitoses.
  11. A single-cell transcriptomic map of the developing Atoh1 lineage identifies neural fate decisions and neuronal diversity in the hindbrain.
  12. Engineered nascent living human tissues with unit programmability.
  13. SLC25A48 controls mitochondrial choline import and metabolism.
  14. Bulk synthesis and beyond: The roles of eukaryotic replicative DNA polymerases.
  15. Cardiomyocyte senescence and the potential therapeutic role of senolytics in the heart.
  16. In situ assembly of an injectable cardiac stimulator.
  17. A developmental mechanism to regulate alternative polyadenylation in an adult stem cell lineage.
  18. Akt is a mediator of artery specification during zebrafish development.
  19. Titration of RAS alters senescent state and influences tumour initiation.
  20. Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single-cell RNA-seq.
  21. Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms.
  22. Nucleocytoplasmic transport senses mechanics independently of cell density in cell monolayers.
  23. PERK-ATAD3A interaction provides a subcellular safe haven for protein synthesis during ER stress.
  24. The ribosomal protein L22 binds the MDM4 pre-mRNA and promotes exon skipping to activate p53 upon nucleolar stress.
  25. Actin networks modulate heterogenous NF-κB dynamics in response to TNFα.
  26. Macrophage OTUD1-CARD9 axis drives isoproterenol-induced inflammatory heart remodelling.
  27. PI4P-mediated solid-like Merlin condensates orchestrate Hippo pathway regulation.
  28. ILC2-derived LIF licences progress from tissue to systemic immunity.
  29. Nesprin-2 coordinates opposing microtubule motors during nuclear migration in neurons.
  1. Dev Cell. 2024 Aug 01. pii: S1534-5807(24)00449-0. [Epub ahead of print]
    Hox gene activity directs physical forces to differentially shape chick small and large intestinal epithelia.
    Hasreet K Gill, Sifan Yin, Nandan L Nerurkar, John C Lawlor, ChangHee Lee, Tyler R Huycke, L Mahadevan, Clifford J Tabin.
      Hox transcription factors play crucial roles in organizing developmental patterning across metazoa, but how these factors trigger regional morphogenesis has largely remained a mystery. In the developing gut, Hox genes help demarcate identities of intestinal subregions early in embryogenesis, which ultimately leads to their specialization in both form and function. Although the midgut forms villi, the hindgut develops sulci that resolve into heterogeneous outgrowths. Combining mechanical measurements of the embryonic chick intestine and mathematical modeling, we demonstrate that the posterior Hox gene HOXD13 regulates biophysical phenomena that shape the hindgut lumen. We further show that HOXD13 acts through the transforming growth factor β (TGF-β) pathway to thicken, stiffen, and promote isotropic growth of the subepithelial mesenchyme-together, these features lead to hindgut-specific surface buckling. TGF-β, in turn, promotes collagen deposition to affect mesenchymal geometry and growth. We thus identify a cascade of events downstream of positional identity that direct posterior intestinal morphogenesis.
    Keywords:  Hox genes; TGF-β pathway; gut; mechanical forces; modeling; morphogenesis
    DOI:  https://doi.org/10.1016/j.devcel.2024.07.012
  2. Cell Genom. 2024 Aug 02. pii: S2666-979X(24)00233-7. [Epub ahead of print] 100629
    Diversity of ribosomes at the level of rRNA variation associated with human health and disease.
    Daphna Rothschild, Teodorus Theo Susanto, Xin Sui, Jeffrey P Spence, Ramya Rangan, Naomi R Genuth, Nasa Sinnott-Armstrong, Xiao Wang, Jonathan K Pritchard, Maria Barna.
      With hundreds of copies of rDNA, it is unknown whether they possess sequence variations that form different types of ribosomes. Here, we developed an algorithm for long-read variant calling, termed RGA, which revealed that variations in human rDNA loci are predominantly insertion-deletion (indel) variants. We developed full-length rRNA sequencing (RIBO-RT) and in situ sequencing (SWITCH-seq), which showed that translating ribosomes possess variation in rRNA. Over 1,000 variants are lowly expressed. However, tens of variants are abundant and form distinct rRNA subtypes with different structures near indels as revealed by long-read rRNA structure probing coupled to dimethyl sulfate sequencing. rRNA subtypes show differential expression in endoderm/ectoderm-derived tissues, and in cancer, low-abundance rRNA variants can become highly expressed. Together, this study identifies the diversity of ribosomes at the level of rRNA variants, their chromosomal location, and unique structure as well as the association of ribosome variation with tissue-specific biology and cancer.
    Keywords:  in situ sequencing; rDNA; rDNA sequence variations; rRNA; rRNA association with development and cancer; rRNA sequence variations; rRNA structure; repetitive genomic elements; ribosome; ribosome heterogeneity
    DOI:  https://doi.org/10.1016/j.xgen.2024.100629
  3. Dev Cell. 2024 Jul 30. pii: S1534-5807(24)00445-3. [Epub ahead of print]
    Redox heterogeneity in mouse embryonic stem cells individualizes cell fate decisions.
    Agnes Ulfig, Ursula Jakob.
      Pluripotent embryonic stem cells (ESCs) can develop into any cell type in the body. Yet, the regulatory mechanisms that govern cell fate decisions during embryogenesis remain largely unknown. We now demonstrate that mouse ESCs (mESCs) display large natural variations in mitochondrial reactive oxygen species (mitoROS) levels that individualize their nuclear redox state, H3K4me3 landscape, and cell fate. While mESCs with high mitoROS levels (mitoROSHIGH) differentiate toward mesendoderm and form the primitive streak during gastrulation, mESCs, which generate less ROS, choose the alternative neuroectodermal fate. Temporal studies demonstrated that mesendodermal (ME) specification of mitoROSHIGH mESCs is mediated by a Nrf2-controlled switch in the nuclear redox state, triggered by the accumulation of redox-sensitive H3K4me3 marks, and executed by a hitherto unknown ROS-dependent activation process of the Wnt signaling pathway. In summary, our study explains how ESC heterogeneity is generated and used by individual cells to decide between distinct cellular fates.
    Keywords:  H3K4me3 epigenome; Nrf2 signaling; Wnt signaling pathway; cell fate decision; embryonic stem cells; mesendoderm; neuroectoderm; reactive oxygen species; redox; stem cell heterogeneity
    DOI:  https://doi.org/10.1016/j.devcel.2024.07.008
  4. Cell Genom. 2024 Aug 02. pii: S2666-979X(24)00232-5. [Epub ahead of print] 100628
    Stable and robust Xi and Y transcriptomes drive cell-type-specific autosomal and Xa responses in vivo and in vitro in four human cell types.
    Laura V Blanton, Adrianna K San Roman, Geryl Wood, Ashley Buscetta, Nicole Banks, Helen Skaletsky, Alexander K Godfrey, Thao T Pham, Jennifer F Hughes, Laura G Brown, Paul Kruszka, Angela E Lin, Daniel L Kastner, Maximilian Muenke, David C Page.
      Recent in vitro studies of human sex chromosome aneuploidy showed that the Xi ("inactive" X) and Y chromosomes broadly modulate autosomal and Xa ("active" X) gene expression. We tested these findings in vivo. Linear modeling of CD4+ T cells and monocytes from individuals with one to three X chromosomes and zero to two Y chromosomes revealed 82 sex-chromosomal and 344 autosomal genes whose expression changed significantly with Xi and/or Y dosage in vivo. Changes in sex-chromosomal expression were remarkably constant in vivo and in vitro; autosomal responses to Xi and/or Y dosage were largely cell-type specific (∼2.6-fold more variation than sex-chromosomal responses). Targets of the sex-chromosomal transcription factors ZFX and ZFY accounted for a significant fraction of these autosomal responses both in vivo and in vitro. We conclude that the human Xi and Y transcriptomes are surprisingly robust and stable, yet they modulate autosomal and Xa genes in a cell-type-specific fashion.
    Keywords:  CD4(+) T cell activation; aneuploidy; gene expression; linear modeling; primary CD4(+) T cells; primary monocytes; sex chromosomes; sex differences
    DOI:  https://doi.org/10.1016/j.xgen.2024.100628
  5. Nat Aging. 2024 Aug 08.
    Age-associated clonal B cells drive B cell lymphoma in mice.
    José P Castro, Anastasia V Shindyapina, Alessandro Barbieri, Kejun Ying, Olga S Strelkova, João A Paulo, Alexander Tyshkovskiy, Rico Meinl, Csaba Kerepesi, Anna P Petrashen, Marco Mariotti, Margarita V Meer, Yan Hu, Alexander Karamyshev, Grigoriy Losyev, Mafalda Galhardo, Elsa Logarinho, Artur A Indzhykulian, Steven P Gygi, John M Sedivy, John P Manis, Vadim N Gladyshev.
      Although cancer is an age-related disease, how the processes of aging contribute to cancer progression is not well understood. In this study, we uncovered how mouse B cell lymphoma develops as a consequence of a naturally aged system. We show here that this malignancy is associated with an age-associated clonal B cell (ACBC) population that likely originates from age-associated B cells. Driven by c-Myc activation, promoter hypermethylation and somatic mutations, IgM+ ACBCs clonally expand independently of germinal centers and show increased biological age. ACBCs become self-sufficient and support malignancy when transferred into young recipients. Inhibition of mTOR or c-Myc in old mice attenuates pre-malignant changes in B cells during aging. Although the etiology of mouse and human B cell lymphomas is considered distinct, epigenetic changes in transformed mouse B cells are enriched for changes observed in human B cell lymphomas. Together, our findings characterize the spontaneous progression of cancer during aging through both cell-intrinsic and microenvironmental changes and suggest interventions for its prevention.
    DOI:  https://doi.org/10.1038/s43587-024-00671-7
  6. Nat Cell Biol. 2024 Aug 05.
    p16-dependent increase of PD-L1 stability regulates immunosurveillance of senescent cells.
    Julia Majewska, Amit Agrawal, Avi Mayo, Lior Roitman, Rishita Chatterjee, Jarmila Sekeresova Kralova, Tomer Landsberger, Yonatan Katzenelenbogen, Tomer Meir-Salame, Efrat Hagai, Ilanit Sopher, Juan-Felipe Perez-Correa, Wolfgang Wagner, Avi Maimon, Ido Amit, Uri Alon, Valery Krizhanovsky.
      The accumulation of senescent cells promotes ageing and age-related diseases, but molecular mechanisms that senescent cells use to evade immune clearance and accumulate in tissues remain to be elucidated. Here we report that p16-positive senescent cells upregulate the immune checkpoint protein programmed death-ligand 1 (PD-L1) to accumulate in ageing and chronic inflammation. We show that p16-mediated inhibition of cell cycle kinases CDK4/6 induces PD-L1 stability in senescent cells via downregulation of its ubiquitin-dependent degradation. p16-expressing senescent alveolar macrophages elevate PD-L1 to promote an immunosuppressive environment that can contribute to an increased burden of senescent cells. Treatment with activating anti-PD-L1 antibodies engaging Fcγ receptors on effector cells leads to the elimination of PD-L1 and p16-positive cells. Our study uncovers a molecular mechanism of p16-dependent regulation of PD-L1 protein stability in senescent cells and reveals the potential of targeting PD-L1 to improve immunosurveillance of senescent cells and ameliorate senescence-associated inflammation.
    DOI:  https://doi.org/10.1038/s41556-024-01465-0
  7. Nature. 2024 Aug 07.
    A spatial expression atlas of the adult human proximal small intestine.
    Yotam Harnik, Oran Yakubovsky, Rouven Hoefflin, Roy Novoselsky, Keren Bahar Halpern, Tal Barkai, Yael Korem Kohanim, Adi Egozi, Ofra Golani, Yoseph Addadi, Merav Kedmi, Tal Keidar Haran, Yishai Levin, Alon Savidor, Hadas Keren-Shaul, Chen Mayer, Niv Pencovich, Ron Pery, Dror S Shouval, Itay Tirosh, Ido Nachmany, Shalev Itzkovitz.
      The mouse small intestine shows profound variability in gene expression along the crypt-villus axis1,2. Whether similar spatial heterogeneity exists in the adult human gut remains unclear. Here we use spatial transcriptomics, spatial proteomics and single-molecule fluorescence in situ hybridization to reconstruct a comprehensive spatial expression atlas of the adult human proximal small intestine. We describe zonated expression and cell type representation for epithelial, mesenchymal and immune cell types. We find that migrating enterocytes switch from lipid droplet assembly and iron uptake at the villus bottom to chylomicron biosynthesis and iron release at the tip. Villus tip cells are pro-immunogenic, recruiting γδ T cells and macrophages to the tip, in contrast to their immunosuppressive roles in mouse. We also show that the human small intestine contains abundant serrated and branched villi that are enriched at the tops of circular folds. Our study presents a detailed resource for understanding the biology of the adult human small intestine.
    DOI:  https://doi.org/10.1038/s41586-024-07793-3
  8. Sci Adv. 2024 Aug 09. 10(32): eadp0860
    Active shape programming drives Drosophila wing disc eversion.
    Jana F Fuhrmann, Abhijeet Krishna, Joris Paijmans, Charlie Duclut, Greta Cwikla, Suzanne Eaton, Marko Popović, Frank Jülicher, Carl D Modes, Natalie A Dye.
      How complex 3D tissue shape emerges during animal development remains an important open question in biology and biophysics. Here, we discover a mechanism for 3D epithelial shape change based on active, in-plane cellular events that is analogous to inanimate "shape programmable" materials, which undergo blueprinted 3D shape transformations from in-plane gradients of spontaneous strains. We study eversion of the Drosophila wing disc pouch, when the epithelium transforms from a dome into a curved fold, quantifying 3D tissue shape changes and mapping spatial patterns of cellular behaviors on the evolving geometry using cellular topology. Using a physical model inspired by shape programming, we find that active cell rearrangements are the major contributor to pouch eversion and validate this conclusion using a knockdown of MyoVI, which reduces rearrangements and disrupts morphogenesis. This work shows that shape programming is a mechanism for animal tissue morphogenesis and suggests that patterns in nature could present design strategies for shape-programmable materials.
    DOI:  https://doi.org/10.1126/sciadv.adp0860
  9. Nature. 2024 Aug 07.
    Membrane prewetting by condensates promotes tight-junction belt formation.
    Karina Pombo-García, Omar Adame-Arana, Cecilie Martin-Lemaitre, Frank Jülicher, Alf Honigmann.
      Biomolecular condensates enable cell compartmentalization by acting as membraneless organelles1. How cells control the interactions of condensates with other cellular structures such as membranes to drive morphological transitions remains poorly understood. We discovered that formation of a tight-junction belt, which is essential for sealing epithelial tissues, is driven by a wetting phenomenon that promotes the growth of a condensed ZO-1 layer2 around the apical membrane interface. Using temporal proximity proteomics in combination with imaging and thermodynamic theory, we found that the polarity protein PATJ mediates a transition of ZO-1 into a condensed surface layer that elongates around the apical interface. In line with the experimental observations, our theory of condensate growth shows that the speed of elongation depends on the binding affinity of ZO-1 to the apical interface and is constant. Here, using PATJ mutations, we show that ZO-1 interface binding is necessary and sufficient for tight-junction belt formation. Our results demonstrate how cells exploit the collective biophysical properties of protein condensates at membrane interfaces to shape mesoscale structures.
    DOI:  https://doi.org/10.1038/s41586-024-07726-0
  10. J Cell Biol. 2024 Nov 04. pii: e202308034. [Epub ahead of print]223(11):
    Cyclin B3 is a dominant fast-acting cyclin that drives rapid early embryonic mitoses.
    Pablo Lara-Gonzalez, Smriti Variyar, Shabnam Moghareh, Anh Cao Ngoc Nguyen, Amrutha Kizhedathu, Jacqueline Budrewicz, Aleesa Schlientz, Neha Varshney, Andrew Bellaart, Karen Oegema, Lee Bardwell, Arshad Desai.
      Mitosis in early embryos often proceeds at a rapid pace, but how this pace is achieved is not understood. Here, we show that cyclin B3 is the dominant driver of rapid embryonic mitoses in the C. elegans embryo. Cyclins B1 and B2 support slow mitosis (NEBD to anaphase ∼600 s), but the presence of cyclin B3 dominantly drives the approximately threefold faster mitosis observed in wildtype. Multiple mitotic events are slowed down in cyclin B1 and B2-driven mitosis, and cyclin B3-associated Cdk1 H1 kinase activity is ∼25-fold more active than cyclin B1-associated Cdk1. Addition of cyclin B1 to fast cyclin B3-only mitosis introduces an ∼60-s delay between completion of chromosome alignment and anaphase onset; this delay, which is important for segregation fidelity, is dependent on inhibitory phosphorylation of the anaphase activator Cdc20. Thus, cyclin B3 dominance, coupled to a cyclin B1-dependent delay that acts via Cdc20 phosphorylation, sets the rapid pace and ensures mitotic fidelity in the early C. elegans embryo.
    DOI:  https://doi.org/10.1083/jcb.202308034
  11. Dev Cell. 2024 Jul 31. pii: S1534-5807(24)00443-X. [Epub ahead of print]
    A single-cell transcriptomic map of the developing Atoh1 lineage identifies neural fate decisions and neuronal diversity in the hindbrain.
    Jessica C Butts, Sih-Rong Wu, Mark A Durham, Ryan S Dhindsa, Jean-Pierre Revelli, M Cecilia Ljungberg, Olivier Saulnier, Madison E McLaren, Michael D Taylor, Huda Y Zoghbi.
      Proneural transcription factors establish molecular cascades to orchestrate neuronal diversity. One such transcription factor, Atonal homolog 1 (Atoh1), gives rise to cerebellar excitatory neurons and over 30 distinct nuclei in the brainstem critical for hearing, breathing, and balance. Although Atoh1 lineage neurons have been qualitatively described, the transcriptional programs that drive their fate decisions and the full extent of their diversity remain unknown. Here, we analyzed single-cell RNA sequencing and ATOH1 DNA binding in Atoh1 lineage neurons of the developing mouse hindbrain. This high-resolution dataset identified markers for specific brainstem nuclei and demonstrated that transcriptionally heterogeneous progenitors require ATOH1 for proper migration. Moreover, we identified a sizable population of proliferating unipolar brush cell progenitors in the mouse Atoh1 lineage, previously described in humans as the origin of one medulloblastoma subtype. Collectively, our data provide insights into the developing mouse hindbrain and markers for functional assessment of understudied neuronal populations.
    Keywords:  Atoh1; CUT&RUN; DNA binding; brainstem; cerebellum; hindbrain; neural fate decisions; single-cell RNA sequencing; spatial transcriptomics
    DOI:  https://doi.org/10.1016/j.devcel.2024.07.007
  12. Nat Mater. 2024 Aug 08.
    Engineered nascent living human tissues with unit programmability.
    Pedro Lavrador, Beatriz S Moura, José Almeida-Pinto, Vítor M Gaspar, João F Mano.
      Leveraging human cells as materials precursors is a promising approach for fabricating living materials with tissue-like functionalities and cellular programmability. Here we describe a set of cellular units with metabolically engineered glycoproteins that allow cells to tether together to function as macrotissue building blocks and bioeffectors. The generated human living materials, termed as Cellgels, can be rapidly assembled in a wide variety of programmable three-dimensional configurations with physiologically relevant cell densities (up to 108 cells per cm3), tunable mechanical properties and handleability. Cellgels inherit the ability of living cells to sense and respond to their environment, showing autonomous tissue-integrative behaviour, mechanical maturation, biological self-healing, biospecific adhesion and capacity to promote wound healing. These living features also enable the modular bottom-up assembly of multiscale constructs, which are reminiscent of human tissue interfaces with heterogeneous composition. This technology can potentially be extended to any human cell type, unlocking the possibility for fabricating living materials that harness the intrinsic biofunctionalities of biological systems.
    DOI:  https://doi.org/10.1038/s41563-024-01958-1
  13. Cell Metab. 2024 Aug 01. pii: S1550-4131(24)00281-X. [Epub ahead of print]
    SLC25A48 controls mitochondrial choline import and metabolism.
    Anthony R P Verkerke, Xu Shi, Mark Li, Yusuke Higuchi, Tadashi Yamamuro, Daisuke Katoh, Hiroshi Nishida, Christopher Auger, Ichitaro Abe, Robert E Gerszten, Shingo Kajimura.
      Choline is an essential nutrient for the biosynthesis of phospholipids, neurotransmitters, and one-carbon metabolism with a critical step being its import into mitochondria. However, the underlying mechanisms and biological significance remain poorly understood. Here, we report that SLC25A48, a previously uncharacterized mitochondrial inner-membrane carrier protein, controls mitochondrial choline transport and the synthesis of choline-derived methyl donors. We found that SLC25A48 was required for brown fat thermogenesis, mitochondrial respiration, and mitochondrial membrane integrity. Choline uptake into the mitochondrial matrix via SLC25A48 facilitated the synthesis of betaine and purine nucleotides, whereas loss of SLC25A48 resulted in increased production of mitochondrial reactive oxygen species and imbalanced mitochondrial lipids. Notably, human cells carrying a single nucleotide polymorphism on the SLC25A48 gene and cancer cells lacking SLC25A48 exhibited decreased mitochondrial choline import, increased oxidative stress, and impaired cell proliferation. Together, this study demonstrates that SLC25A48 regulates mitochondrial choline catabolism, bioenergetics, and cell survival.
    Keywords:  bioenergetics; brown adipose tissue; cancer metabolism; choline; mitochondria; purine nucleotides
    DOI:  https://doi.org/10.1016/j.cmet.2024.07.010
  14. DNA Repair (Amst). 2024 Jul 30. pii: S1568-7864(24)00116-2. [Epub ahead of print]141 103740
    Bulk synthesis and beyond: The roles of eukaryotic replicative DNA polymerases.
    Lewis J Bainbridge, Yasukazu Daigaku.
      An organism's genomic DNA must be accurately duplicated during each cell cycle. DNA synthesis is catalysed by DNA polymerase enzymes, which extend nucleotide polymers in a 5' to 3' direction. This inherent directionality necessitates that one strand is synthesised forwards (leading), while the other is synthesised backwards discontinuously (lagging) to couple synthesis to the unwinding of duplex DNA. Eukaryotic cells possess many diverse polymerases that coordinate to replicate DNA, with the three main replicative polymerases being Pol α, Pol δ and Pol ε. Studies conducted in yeasts and human cells utilising mutant polymerases that incorporate molecular signatures into nascent DNA implicate Pol ε in leading strand synthesis and Pol α and Pol δ in lagging strand replication. Recent structural insights have revealed how the spatial organization of these enzymes around the core helicase facilitates their strand-specific roles. However, various challenging situations during replication require flexibility in the usage of these enzymes, such as during replication initiation or encounters with replication-blocking adducts. This review summarises the roles of the replicative polymerases in bulk DNA replication and explores their flexible and dynamic deployment to complete genome replication. We also examine how polymerase usage patterns can inform our understanding of global replication dynamics by revealing replication fork directionality to identify regions of replication initiation and termination.
    Keywords:  DNA polymerase; DNA replication; Replisome
    DOI:  https://doi.org/10.1016/j.dnarep.2024.103740
  15. J Cardiovasc Aging. 2024 Apr;pii: 18. [Epub ahead of print]4(2):
    Cardiomyocyte senescence and the potential therapeutic role of senolytics in the heart.
    Peiyong Zhai, Junichi Sadoshima.
      Cellular senescence in cardiomyocytes, characterized by cell cycle arrest, resistance to apoptosis, and the senescence-associated secretory phenotype, occurs during aging and in response to various stresses, such as hypoxia/reoxygenation, ischemia/reperfusion, myocardial infarction (MI), pressure overload, doxorubicin treatment, angiotensin II, diabetes, and thoracic irradiation. Senescence in the heart has both beneficial and detrimental effects. Premature senescence of myofibroblasts has salutary effects during MI and pressure overload. On the other hand, persistent activation of senescence in cardiomyocytes precipitates cardiac dysfunction and adverse remodeling through paracrine mechanisms during MI, myocardial ischemia/reperfusion, aging, and doxorubicin-induced cardiomyopathy. Given the adverse roles of senescence in many conditions, specific removal of senescent cells, i.e., senolysis, is of great interest. Senolysis can be achieved using senolytic drugs (such as Navitoclax, Dasatinib, and Quercetin), pharmacogenetic approaches (including INK-ATTAC and AP20187, p16-3MR and Ganciclovir, p16 ablation, and p16-LOX-ATTAC and Cre), and immunogenetic interventions (CAR T cells or senolytic vaccination). In order to enhance the specificity and decrease the off-target effects of senolytic approaches, investigation into the mechanisms through which cardiomyocytes develop and/or maintain the senescent state is needed.
    Keywords:  Aging; senescence; senescence-associated secretory phenotype; senolysis
    DOI:  https://doi.org/10.20517/jca.2024.06
  16. Nat Commun. 2024 Aug 08. 15(1): 6774
    In situ assembly of an injectable cardiac stimulator.
    Umut Aydemir, Abdelrazek H Mousa, Cedric Dicko, Xenofon Strakosas, Muhammad Anwar Shameem, Karin Hellman, Amit Singh Yadav, Peter Ekström, Damien Hughes, Fredrik Ek, Magnus Berggren, Anders Arner, Martin Hjort, Roger Olsson.
      Without intervention, cardiac arrhythmias pose a risk of fatality. However, timely intervention can be challenging in environments where transporting a large, heavy defibrillator is impractical, or emergency surgery to implant cardiac stimulation devices is not feasible. Here, we introduce an injectable cardiac stimulator, a syringe loaded with a nanoparticle solution comprising a conductive polymer and a monomer that, upon injection, forms a conductive structure around the heart for cardiac stimulation. Following treatment, the electrode is cleared from the body, eliminating the need for surgical extraction. The mixture adheres to the beating heart in vivo without disrupting its normal rhythm. The electrofunctionalized injectable cardiac stimulator demonstrates a tissue-compatible Young's modulus of 21 kPa and a high conductivity of 55 S/cm. The injected electrode facilitates electrocardiogram measurements, regulates heartbeat in vivo, and rectifies arrhythmia. Conductive functionality is maintained for five consecutive days, and no toxicity is observed at the organism, organ, or cellular levels.
    DOI:  https://doi.org/10.1038/s41467-024-51111-4
  17. Genes Dev. 2024 Aug 07.
    A developmental mechanism to regulate alternative polyadenylation in an adult stem cell lineage.
    Lorenzo Gallicchio, Neuza R Matias, Fábian Morales-Polanco, Iliana Nava, Sarah Stern, Yi Zeng, Margaret T Fuller.
      Alternative cleavage and polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3' UTRs from the same genetic locus, potentially impacting mRNA translation, localization, and stability. Developmentally regulated APA can thus make major contributions to cell type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, ∼500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3' UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of cleavage factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knockdown of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell type-specific APA at selected genes.
    Keywords:  Drosophila; alternative polyadenylation; cellular differentiation; cleavage factor complex II; development; mRNA processing; spermatogenesis
    DOI:  https://doi.org/10.1101/gad.351649.124
  18. Development. 2024 Aug 05. pii: dev.202727. [Epub ahead of print]
    Akt is a mediator of artery specification during zebrafish development.
    Wenping Zhou, Joey J Ghersi, Emma Ristori, Nicole Semanchik, Andrew Prendergast, Rong Zhang, Paola Carneiro, Gabriel Baldissera, William C Sessa, Stefania Nicoli.
      The dorsal aorta (DA) is the first major blood vessel to develop in the embryonic cardiovascular system. Its formation is governed by a coordinated process involving the migration, specification, and arrangement of angioblasts into arterial and venous lineages, a process conserved across species. While vascular endothelial growth factor a (VEGF-A) drives DA specification and formation, the kinases involved in this process remain ambiguous. Thus, we investigated the role of protein kinase B, Akt, in zebrafish by generating a quadruple mutant (aktΔ/Δ), where expression and activity of all akt genes-akt 1, 2, 3a, and 3b are strongly decreased. Live imaging of developing aktΔ/Δ DA uncovers early arteriovenous malformations. Single-cell RNA sequencing analysis of aktΔ/Δ endothelial cells corroborates the impairment of arterial, yet not venous, cell specification. Notably, endothelial specific expression of ligand-independent activation of Notch or constitutively active Akt1 were sufficient to reestablish normal arterial specification in aktΔ/Δ. The Akt-loss-of-function mutant unveils that Akt kinase can act upstream of Notch in arterial endothelial cells, and is involved in proper embryonic artery specification. This sheds light on cardiovascular development, revealing a mechanism behind congenital malformations.
    Keywords:  Akt and Notch signaling; Artery specification; Endothelial cells; Single cell RNA sequencing.; Vascular development; Zebrafish embryo
    DOI:  https://doi.org/10.1242/dev.202727
  19. Nature. 2024 Aug 07.
    Titration of RAS alters senescent state and influences tumour initiation.
    Adelyne S L Chan, Haoran Zhu, Masako Narita, Liam D Cassidy, Andrew R J Young, Camino Bermejo-Rodriguez, Aleksandra T Janowska, Hung-Chang Chen, Sarah Gough, Naoki Oshimori, Lars Zender, Sarah J Aitken, Matthew Hoare, Masashi Narita.
      Oncogenic RAS-induced senescence (OIS) is an autonomous tumour suppressor mechanism associated with premalignancy1,2. Achieving this phenotype typically requires a high level of oncogenic stress, yet the phenotype provoked by lower oncogenic dosage remains unclear. Here we develop oncogenic RAS dose-escalation models in vitro and in vivo, revealing a RAS dose-driven non-linear continuum of downstream phenotypes. In a hepatocyte OIS model in vivo, ectopic expression of NRAS(G12V) does not induce tumours, in part owing to OIS-driven immune clearance3. Single-cell RNA sequencing analyses reveal distinct hepatocyte clusters with typical OIS or progenitor-like features, corresponding to high and intermediate levels of NRAS(G12V), respectively. When titred down, NRAS(G12V)-expressing hepatocytes become immune resistant and develop tumours. Time-series monitoring at single-cell resolution identifies two distinct tumour types: early-onset aggressive undifferentiated and late-onset differentiated hepatocellular carcinoma. The molecular signature of each mouse tumour type is associated with different progenitor features and enriched in distinct human hepatocellular carcinoma subclasses. Our results define the oncogenic dosage-driven OIS spectrum, reconciling the senescence and tumour initiation phenotypes in early tumorigenesis.
    DOI:  https://doi.org/10.1038/s41586-024-07797-z
  20. Cell Rep. 2024 Aug 04. pii: S2211-1247(24)00922-7. [Epub ahead of print]43(8): 114593
    Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single-cell RNA-seq.
    Jeremy A Schofield, Steven Hahn.
      We describe a time-resolved nascent single-cell RNA sequencing (RNA-seq) approach that measures gene-specific transcriptional noise and the fraction of active genes in S. cerevisiae. Most genes are expressed with near-constitutive behavior, while a subset of genes show high mRNA variance suggestive of transcription bursting. Transcriptional noise is highest in the cofactor/coactivator-redundant (CR) gene class (dependent on both SAGA and TFIID) and strongest in TATA-containing CR genes. Using this approach, we also find that histone gene transcription switches from a low-level, low-noise constitutive mode during M and M/G1 to an activated state in S phase that shows both an increase in the fraction of active promoters and a switch to a noisy and bursty transcription mode. Rapid depletion of cofactors SAGA and MED Tail indicates that both factors play an important role in stimulating the fraction of active promoters at CR genes, with a more modest role in transcriptional noise.
    Keywords:  CP: Molecular biology; Mediator; S. cerevisiae; SAGA; bursting; gene activation; single-cell RNA-seq; transcription; transcription noise; yeast
    DOI:  https://doi.org/10.1016/j.celrep.2024.114593
  21. Nat Commun. 2024 Aug 08. 15(1): 6777
    Metabolic and transcriptomic reprogramming during contact inhibition-induced quiescence is mediated by YAP-dependent and YAP-independent mechanisms.
    Soeun Kang, Maciek R Antoniewicz, Nissim Hay.
      Metabolic rewiring during the proliferation-to-quiescence transition is poorly understood. Here, using a model of contact inhibition-induced quiescence, we conducted 13C-metabolic flux analysis in proliferating (P) and quiescent (Q) mouse embryonic fibroblasts (MEFs) to investigate this process. Q cells exhibit reduced glycolysis but increased TCA cycle flux and mitochondrial respiration. Reduced glycolytic flux in Q cells correlates with reduced glycolytic enzyme expression mediated by yes-associated protein (YAP) inhibition. The increased TCA cycle activity and respiration in Q cells is mediated by induced mitochondrial pyruvate carrier (MPC) expression, rendering them vulnerable to MPC inhibition. The malate-to-pyruvate flux, which generates NADPH, is markedly reduced by modulating malic enzyme 1 (ME1) dimerization in Q cells. Conversely, the malate dehydrogenase 1 (MDH1)-mediated oxaloacetate-to-malate flux is reversed and elevated in Q cells, driven by high mitochondrial-derived malate levels, reduced cytosolic oxaloacetate, elevated MDH1 levels, and a high cytoplasmic NAD+/NADH ratio. Transcriptomic analysis revealed large number of genes are induced in Q cells, many of which are associated with the extracellular matrix (ECM), while YAP-dependent and cell cycle-related genes are repressed. The results suggest that high TCA cycle flux and respiration in Q cells are required to generate ATP and amino acids to maintain de-novo ECM protein synthesis and secretion.
    DOI:  https://doi.org/10.1038/s41467-024-51117-y
  22. J Cell Sci. 2024 Aug 09. pii: jcs.262363. [Epub ahead of print]
    Nucleocytoplasmic transport senses mechanics independently of cell density in cell monolayers.
    Ignasi Granero-Moya, Valeria Venturini, Guillaume Belthier, Bart Groenen, Marc Molina-Jordán, Miguel González-Martín, Xavier Trepat, Jacco van Rheenen, Ion Andreu, Pere Roca-Cusachs.
      Cells sense and respond to mechanical forces through mechanotransduction, which regulates processes in health and disease. In single adhesive cells, mechanotransduction involves the transmission of force from the extracellular matrix to the cell nucleus, where it affects nucleocytoplasmic transport (NCT) and the subsequent nuclear localization of transcriptional regulators such as YAP. However, if and how NCT is mechanosensitive in multicellular systems is unclear. Here, we characterize and use a fluorescent sensor of nucleocytoplasmic transport (Sencyt) and demonstrate that nucleocytoplasmic transport responds to mechanics but not cell density in cell monolayers. Using monolayers of both epithelial and mesenchymal phenotype, we show that NCT is altered in response both to osmotic shocks, and to the inhibition of cell contractility. Further, NCT correlates with the degree of nuclear deformation measured through nuclear solidity, a shape parameter related to nuclear envelope tension. In contrast, YAP but NCT is sensitive to cell density, showing that YAP response to cell-cell contacts is not via a mere mechanical effect of NCT. Our results demonstrate the generality of the mechanical regulation of NCT.
    Keywords:  Cell nucleus; Mechanobiology; Mechanotransduction; Sensor
    DOI:  https://doi.org/10.1242/jcs.262363
  23. Science. 2024 Aug 08. eadp7114
    PERK-ATAD3A interaction provides a subcellular safe haven for protein synthesis during ER stress.
    Karinder K Brar, Daniel T Hughes, Jordan L Morris, Kelly Subramanian, Shivaani Krishna, Fei Gao, Lara-Sophie Rieder, Sebastian Uhrig, Joshua Freeman, Heather L Smith, Rebekkah Jukes-Jones, Edward Avezov, Jodi Nunnari, Julien Prudent, Adrian J Butcher, Giovanna R Mallucci.
      Endoplasmic Reticulum (ER) stress induces repression of protein synthesis throughout the cell. Attempts to understand how localized stress leads to widespread repression have been limited by difficulties in resolving translation rates at the subcellular level. Here, using live-cell imaging of reporter mRNA translation, we unexpectedly found that during ER stress active translation at mitochondria was significantly protected. The mitochondrial protein, ATAD3A, interacted with PERK and mediated this effect on localized translation by competing for binding with PERK's target, eIF2. PERK-ATAD3A interactions increased during ER stress, forming mitochondria-ER contact sites. Furthermore, ATAD3A binding attenuated local PERK signaling and rescued the expression of some mitochondrial proteins. Thus, PERK-ATAD3A interactions can control translational repression at a subcellular level, mitigating the impact of ER stress on the cell.
    DOI:  https://doi.org/10.1126/science.adp7114
  24. Cell Rep. 2024 Aug 06. pii: S2211-1247(24)00960-4. [Epub ahead of print]43(8): 114610
    The ribosomal protein L22 binds the MDM4 pre-mRNA and promotes exon skipping to activate p53 upon nucleolar stress.
    Jennifer Jansen, Katherine E Bohnsack, Susanne Böhlken-Fascher, Markus T Bohnsack, Matthias Dobbelstein.
      The tumor suppressor p53 and its antagonists MDM2 and MDM4 integrate stress signaling. For instance, dysbalanced assembly of ribosomes in nucleoli induces p53. Here, we show that the ribosomal protein L22 (RPL22; eL22), under conditions of ribosomal and nucleolar stress, promotes the skipping of MDM4 exon 6. Upon L22 depletion, more full-length MDM4 is maintained, leading to diminished p53 activity and enhanced cellular proliferation. L22 binds to specific RNA elements within intron 6 of MDM4 that correspond to a stem-loop consensus, leading to exon 6 skipping. Targeted deletion of these intronic elements largely abolishes L22-mediated exon skipping and re-enables cell proliferation, despite nucleolar stress. L22 also governs alternative splicing of the L22L1 (RPL22L1) and UBAP2L mRNAs. Thus, L22 serves as a signaling intermediate that integrates different layers of gene expression. Defects in ribosome synthesis lead to specific alternative splicing, ultimately triggering p53-mediated transcription and arresting cell proliferation.
    Keywords:  (RP)L22; (RP)L22L1; (RP)S13; CP: Molecular biology; RNA polymerase I; RNA stem-loop; UBAP2L; ZMAT3; exon skipping; nucleolus; pre-mRNA splicing; ribosomal proteins
    DOI:  https://doi.org/10.1016/j.celrep.2024.114610
  25. Elife. 2024 Aug 07. pii: e86042. [Epub ahead of print]13
    Actin networks modulate heterogenous NF-κB dynamics in response to TNFα.
    Francesca Butera, Julia E Sero, Lucas G Dent, Chris Bakal.
      The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in PDAC tumours. In this study, we characterised previously unexplored endogenous RELA-GFP dynamics in PDAC cell lines through live single cell imaging. Our observations revealed that TNFα stimulation induces rapid, sustained, and non-oscillatory nuclear translocation of RELA. Through Bayesian analysis of single cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. RNA-seq analysis identified distinct clusters of RELA-regulated gene expression in PDAC cells, including TNFα-induced RELA upregulation of the actin regulators NUAK2 and ARHGAP31. Further, siRNA-mediated depletion of ARHGAP31 and NUAK2 altered TNFα-stimulated nuclear RELA dynamics in PDAC cells, establishing a novel negative feedback loop that regulates RELA activation by TNFα. Additionally, we characterised the NF-κB pathway in PDAC cells, identifying how NF-κB/IκB proteins genetically and physically interact with RELA in the absence or presence of TNFα. Taken together, we provide computational and experimental support for interdependence between the F-actin network and the NF-κB pathway with RELA translocation dynamics in PDAC.
    Keywords:  cancer biology; cell biology; human
    DOI:  https://doi.org/10.7554/eLife.86042
  26. Clin Transl Med. 2024 Aug;14(8): e1790
    Macrophage OTUD1-CARD9 axis drives isoproterenol-induced inflammatory heart remodelling.
    Jinfu Qian, Qinyan Wang, Jiachen Xu, Shiqi Liang, Qingsong Zheng, Xiaocheng Guo, Wu Luo, Weijian Huang, Xiaohong Long, Julian Min, Yi Wang, Gaojun Wu, Guang Liang.
       BACKGROUND: Chronic inflammation contributes to the progression of isoproterenol (ISO)-induced heart failure (HF). Caspase-associated recruitment domain (CARD) families are crucial proteins for initiation of inflammation in innate immunity. Nonetheless, the relevance of CARDs in ISO-driven cardiac remodelling is little explored.
    METHODS: This study utilized Card9-/- mice and reconstituted C57BL/6 mice with either Card9-/- or Otud1-/- marrow-derived cells. Mechanistic studies were conducted in primary macrophages, cardiomyocytes, fibroblasts and HEK-293T cells.
    RESULTS: Here, we demonstrated that CARD9 was substantially upregulated in murine hearts infused with ISO. Either whole-body CARD9 knockout or myeloid-specific CARD9 deletion inhibited ISO-driven murine cardiac inflammation, remodelling and dysfunction. CARD9 deficiency in macrophages prevented ISO-induced inflammation and alleviated remodelling changes in cardiomyocytes and fibroblasts. Mechanistically, we found that ISO enhances the activity of CARD9 by upregulating ovarian tumour deubiquitinase 1 (OTUD1) in macrophages. We further demonstrated that OTUD1 directly binds to the CARD9 and then removes the K33-linked ubiquitin from CARD9 to promote the assembly of the CARD9-BCL10-MALT1 (CBM) complex, without affecting CARD9 stability. The ISO-activated CBM complex results in NF-κB activation and macrophage-based inflammatory gene overproduction, which then enhances cardiomyocyte hypertrophy and fibroblast fibrosis, respectively. Myeloid-specific OTUD1 deletion also attenuated ISO-induced murine cardiac inflammation and remodelling.
    CONCLUSIONS: These results suggested that the OTUD1-CARD9 axis is a new pro-inflammatory signal in ISO-challenged macrophages and targeting this axis has a protective effect against ISO-induced HF.
    KEY POINTS: Macrophage CARD9 was elevated in heart tissues of mice under chronic ISO administration. Either whole-body CARD9 knockout or myeloid-specific CARD9 deficiency protected mice from ISO-induced inflammatory heart remodeling. ISO promoted the assembly of CBM complex and then activated NF-κB signaling in macrophages through OTUD1-mediated deubiquitinating modification. OTUD1 deletion in myeloid cells protected hearts from ISO-induced injuries in mice.
    Keywords:  CARD9; OTUD1; heart failure; inflammation; isoproterenol; macrophage
    DOI:  https://doi.org/10.1002/ctm2.1790
  27. Science. 2024 Aug 09. 385(6709): eadf4478
    PI4P-mediated solid-like Merlin condensates orchestrate Hippo pathway regulation.
    Pengfei Guo, Bing Li, Wei Dong, Huabin Zhou, Li Wang, Ting Su, Christopher Carl, Yonggang Zheng, Yang Hong, Hua Deng, Duojia Pan.
      Despite recent studies implicating liquid-like biomolecular condensates in diverse cellular processes, many biomolecular condensates exist in a solid-like state, and their function and regulation are less understood. We show that the tumor suppressor Merlin, an upstream regulator of the Hippo pathway, localizes to both cell junctions and medial apical cortex in Drosophila epithelia, with the latter forming solid-like condensates that activate Hippo signaling. Merlin condensation required phosphatidylinositol-4-phosphate (PI4P)-mediated plasma membrane targeting and was antagonistically controlled by Pez and cytoskeletal tension through plasma membrane PI4P regulation. The solid-like material properties of Merlin condensates are essential for physiological function and protect the condensates against external perturbations. Collectively, these findings uncover an essential role for solid-like condensates in normal physiology and reveal regulatory mechanisms for their formation and disassembly.
    DOI:  https://doi.org/10.1126/science.adf4478
  28. Nature. 2024 Aug 07.
    ILC2-derived LIF licences progress from tissue to systemic immunity.
    Mayuri Gogoi, Paula A Clark, Ana C F Ferreira, Noe Rodriguez Rodriguez, Morgan Heycock, Michelle Ko, Jane E Murphy, Victor Chen, Shi-Lu Luan, Helen E Jolin, Andrew N J McKenzie.
      Migration and homing of immune cells are critical for immune surveillance. Trafficking is mediated by combinations of adhesion and chemokine receptors that guide immune cells, in response to chemokine signals, to specific locations within tissues and the lymphatic system to support tissue-localized immune reactions and systemic immunity1,2. Here we show that disruption of leukaemia inhibitory factor (LIF) production from group 2 innate lymphoid cells (ILC2s) prevents immune cells leaving the lungs to migrate to the lymph nodes (LNs). In the absence of LIF, viral infection leads to plasmacytoid dendritic cells (pDCs) becoming retained in the lungs where they improve tissue-localized, antiviral immunity, whereas chronic pulmonary allergen challenge leads to marked immune cell accumulation and the formation of tertiary lymphoid structures in the lung. In both cases immune cells fail to migrate to the lymphatics, leading to highly compromised LN reactions. Mechanistically, ILC2-derived LIF induces the production of the chemokine CCL21 from lymphatic endothelial cells lining the pulmonary lymphatic vessels, thus licensing the homing of CCR7+ immune cells (including dendritic cells) to LNs. Consequently, ILC2-derived LIF dictates the egress of immune cells from the lungs to regulate tissue-localized versus systemic immunity and the balance between allergen and viral responsiveness in the lungs.
    DOI:  https://doi.org/10.1038/s41586-024-07746-w
  29. J Cell Biol. 2024 Nov 04. pii: e202405032. [Epub ahead of print]223(11):
    Nesprin-2 coordinates opposing microtubule motors during nuclear migration in neurons.
    Chuying Zhou, You Kure Wu, Fumiyoshi Ishidate, Takahiro K Fujiwara, Mineko Kengaku.
      Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells.
    DOI:  https://doi.org/10.1083/jcb.202405032
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