bims-ginsta Biomed News
on Genome instability
Issue of 2024–10–20
twenty-six papers selected by
Jinrong Hu, National University of Singapore
  1. Notch induces transcription by stimulating release of paused RNA polymerase II.
  2. Afadin mediates cadherin-catenin complex clustering on F-actin linked to cooperative binding and filament curvature.
  3. Radially patterned morphogenesis of murine hair follicle placodes ensures robust epithelial budding.
  4. Ex vivo imaging reveals the spatiotemporal control of ovulation.
  5. The dynamics and functional impact of tRNA repertoires during early embryogenesis in zebrafish.
  6. Hypertrophic cardiomyopathy-associated mutations drive stromal activation via EGFR-mediated paracrine signaling.
  7. Selective utilization of glucose metabolism guides mammalian gastrulation.
  8. Mesodermal fibronectin controls cell shape, polarity, and mechanotransduction in the second heart field during cardiac outflow tract development.
  9. LINE1 and PRC2 control nucleolar organization and repression of the 8C state in human ESCs.
  10. RNA splicing controls organ-wide maturation of postnatal heart in mice.
  11. Zonated Wnt/β-catenin signal-activated cardiomyocytes at the atrioventricular canal promote coronary vessel formation in zebrafish.
  12. Mitophagy as a guardian against cellular aging.
  13. HDAC7 promotes cardiomyocyte proliferation by suppressing Myocyte Enhancer Factor 2.
  14. Kinesin-5/Cut7 C-terminal tail phosphorylation is essential for microtubule sliding force and bipolar mitotic spindle assembly.
  15. Single-cell resolution of intestinal regeneration in pythons without crypts illuminates conserved vertebrate regenerative mechanisms.
  16. Mechanism of polyadenylation-independent RNA polymerase II termination.
  17. The subcortical maternal complex modulates the cell cycle during early mammalian embryogenesis via 14-3-3.
  18. cGAS deficient mice display premature aging associated with de-repression of LINE1 elements and inflammation.
  19. Multiscale drug screening for cardiac fibrosis identifies MD2 as a therapeutic target.
  20. Analysis of Somatic Mutations in Senescent Cells Using Single-Cell Whole-Genome Sequencing.
  21. Integrative omics reveals the metabolic patterns during oocyte growth.
  22. Quantifying constraint in the human mitochondrial genome.
  23. Structural basis of respiratory complex adaptation to cold temperatures.
  24. Emergent order in epithelial sheets by interplay of cell divisions and cell fate regulation.
  25. Ataxin-2 polyglutamine expansions aberrantly sequester TDP-43 ribonucleoprotein condensates disrupting mRNA transport and local translation in neurons.
  26. Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex.
  1. Genes Dev. 2024 Oct 16.
    Notch induces transcription by stimulating release of paused RNA polymerase II.
    Julia M Rogers, Claudia A Mimoso, Benjamin J E Martin, Alexandre P Martin, Jon C Aster, Karen Adelman, Stephen C Blacklow.
      Notch proteins undergo ligand-induced proteolysis to release a nuclear effector that influences a wide range of cellular processes by regulating transcription. Despite years of study, however, how Notch induces the transcription of its target genes remains unclear. Here, we comprehensively examine the response to human Notch1 across a time course of activation using high-resolution genomic assays of chromatin accessibility and nascent RNA production. Our data reveal that Notch induces target gene transcription primarily by releasing paused RNA polymerase II (RNAPII). Moreover, in contrast to prevailing models suggesting that Notch acts by promoting chromatin accessibility, we found that open chromatin was established at Notch-responsive regulatory elements prior to Notch signal induction through SWI/SNF-mediated remodeling. Together, these studies show that the nuclear response to Notch signaling is dictated by the pre-existing chromatin state and RNAPII distribution at the time of signal activation.
    Keywords:  ATAC-seq; NOTCH1; Notch signaling; PRO-seq; SWI/SNF; TT-seq
    DOI:  https://doi.org/10.1101/gad.352108.124
  2. bioRxiv. 2024 Oct 11. pii: 2024.10.08.617332. [Epub ahead of print]
    Afadin mediates cadherin-catenin complex clustering on F-actin linked to cooperative binding and filament curvature.
    Rui Gong, Matthew J Reynolds, Xiaoyu Sun, Gregory M Alushin.
      The E-cadherin-β-catenin-αE-catenin (cadherin-catenin) complex couples the cytoskeletons of neighboring cells at adherens junctions (AJs) to mediate force transmission across epithelia. Mechanical force and auxiliary binding partners converge to stabilize the cadherin-catenin complex's inherently weak binding to actin filaments (F-actin) through unclear mechanisms. Here we show that afadin's coiled-coil (CC) domain and vinculin synergistically enhance the cadherin-catenin complex's F-actin engagement. The cryo-EM structure of an E-cadherin-β-catenin-αE-catenin-vinculin-afadin-CC supra-complex bound to F-actin reveals that afadin-CC bridges adjacent αE-catenin actin-binding domains along the filament, stabilizing flexible αE-catenin segments implicated in mechanical regulation. These cooperative binding contacts promote the formation of supra-complex clusters along F-actin. Additionally, cryo-EM variability analysis links supra-complex binding along individual F-actin strands to nanoscale filament curvature, a deformation mode associated with cytoskeletal forces. Collectively, this work elucidates a mechanistic framework by which vinculin and afadin tune cadherin-catenin complex-cytoskeleton coupling to support AJ function across varying mechanical regimes.
    DOI:  https://doi.org/10.1101/2024.10.08.617332
  3. Dev Cell. 2024 Oct 11. pii: S1534-5807(24)00571-9. [Epub ahead of print]
    Radially patterned morphogenesis of murine hair follicle placodes ensures robust epithelial budding.
    Liliya Leybova, Abhishek Biswas, Rishabh Sharan, Brandon M Trejo, Keunho Kim, Yanilka Soto-Muniz, Rebecca A Jones, Brooke K Phillips, Danelle Devenport.
      The bending of simple cellular sheets into complex three-dimensional (3D) forms requires developmental patterning cues to specify where deformations occur, but how positional information directs morphological change is poorly understood. Here, we investigate how morphogen signaling and cell fate diversification contribute to the morphogenesis of murine hair placodes, in which collective cell movements transform radially symmetric primordia into bilaterally symmetric tubes. Through live imaging and 3D volumetric reconstructions, we demonstrate that Wnt and Shh establish radial patterns of cell fate, cell morphology, and movement within developing placodes. Cell fate diversity at different radial positions provides unique and essential contributions to placode morphogenesis. Further, we show that downstream of radial patterning, gradients of classical cadherin expression are required for efficient epithelial rearrangements. Given that the transformation of epithelial discs into 3D tubes is a common morphological motif used to shape diverse organ primordia, mechanisms of radially patterned morphogenesis are likely highly conserved across evolution.
    Keywords:  Shh; Wnt; adhesion; cadherin; hair follicle; placode; planar cell polarity; radial patterning
    DOI:  https://doi.org/10.1016/j.devcel.2024.09.022
  4. Nat Cell Biol. 2024 Oct 16.
    Ex vivo imaging reveals the spatiotemporal control of ovulation.
    Christopher Thomas, Tabea Lilian Marx, Sarah Mae Penir, Melina Schuh.
      During ovulation, an egg is released from an ovarian follicle, ready for fertilization. Ovulation occurs inside the body, impeding direct studies of its progression. Therefore, the exact mechanisms that control ovulation have remained unclear. Here we devised live imaging methods to study the entire process of ovulation in isolated mouse ovarian follicles. We show that ovulation proceeds through three distinct phases, follicle expansion (I), contraction (II) and rupture (III), culminating in the release of the egg. Follicle expansion is driven by hyaluronic acid secretion and an osmotic gradient-directed fluid influx into the follicle. Then, smooth muscle cells in the outer follicle drive follicle contraction. Follicle rupture begins with stigma formation, followed by the exit of follicular fluid and cumulus cells and the rapid release of the egg. These results establish a mechanistic framework for ovulation, a process of fundamental importance for reproduction.
    DOI:  https://doi.org/10.1038/s41556-024-01524-6
  5. EMBO J. 2024 Oct 14.
    The dynamics and functional impact of tRNA repertoires during early embryogenesis in zebrafish.
    Madalena M Reimão-Pinto, Andrew Behrens, Sergio Forcelloni, Klemens Fröhlich, Selay Kaya, Danny D Nedialkova.
      Embryogenesis entails dramatic shifts in mRNA translation and turnover that reprogram gene expression during cellular proliferation and differentiation. Codon identity modulates mRNA stability during early vertebrate embryogenesis, but how the composition of tRNA pools is matched to translational demand is unknown. By quantitative profiling of tRNA repertoires in zebrafish embryos during the maternal-to-zygotic transition, we show that zygotic tRNA repertoires are established after the onset of gastrulation, succeeding the major wave of zygotic mRNA transcription. Maternal and zygotic tRNA pools are distinct, but their reprogramming does not result in a better match to the codon content of the zygotic transcriptome. Instead, we find that an increase in global translation at gastrulation sensitizes decoding rates to tRNA supply, thus destabilizing maternal mRNAs enriched in slowly translated codons. Translational activation and zygotic tRNA expression temporally coincide with an increase of TORC1 activity at gastrulation, which phosphorylates and inactivates the RNA polymerase III repressor Maf1a/b. Our data indicate that a switch in global translation, rather than tRNA reprogramming, determines the onset of codon-dependent maternal mRNA decay during zebrafish embryogenesis.
    Keywords:  Maternal-to-zygotic Transition; TORC1; Translation Regulation; Zebrafish; tRNA
    DOI:  https://doi.org/10.1038/s44318-024-00265-4
  6. Sci Adv. 2024 Oct 18. 10(42): eadi6927
    Hypertrophic cardiomyopathy-associated mutations drive stromal activation via EGFR-mediated paracrine signaling.
    Jourdan K Ewoldt, Miranda C Wang, Micheal A McLellan, Paige E Cloonan, Anant Chopra, Joshua Gorham, Linqing Li, Daniel M DeLaughter, Xining Gao, Joshua H Lee, Jon A L Willcox, Olivia Layton, Rebeccah J Luu, Christopher N Toepfer, Jeroen Eyckmans, Christine E Seidman, Jonathan G Seidman, Christopher S Chen.
      Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the left ventricular wall, diastolic dysfunction, and fibrosis, and is associated with mutations in genes encoding sarcomere proteins. While in vitro studies have used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study HCM, these models have not examined the multicellular interactions involved in fibrosis. Using engineered cardiac microtissues (CMTs) composed of HCM-causing MYH7-variant hiPSC-CMs and wild-type fibroblasts, we observed cell-cell cross-talk leading to increased collagen deposition, tissue stiffening, and decreased contractility dependent on fibroblast proliferation. hiPSC-CM conditioned media and single-nucleus RNA sequencing data suggested that fibroblast proliferation is mediated by paracrine signals from MYH7-variant cardiomyocytes. Furthermore, inhibiting epidermal growth factor receptor tyrosine kinase with erlotinib hydrochloride attenuated stromal activation. Last, HCM-causing MYBPC3-variant CMTs also demonstrated increased stromal activation and reduced contractility, but with distinct characteristics. Together, these findings establish a paracrine-mediated cross-talk potentially responsible for fibrotic changes observed in HCM.
    DOI:  https://doi.org/10.1126/sciadv.adi6927
  7. Nature. 2024 Oct 16.
    Selective utilization of glucose metabolism guides mammalian gastrulation.
    Dominica Cao, Jenna Bergmann, Liangwen Zhong, Anupama Hemalatha, Chaitanya Dingare, Tyler Jensen, Andy L Cox, Valentina Greco, Benjamin Steventon, Berna Sozen.
      The prevailing dogma for morphological patterning in developing organisms argues that the combined inputs of transcription factor networks and signalling morphogens alone generate spatially and temporally distinct expression patterns. However, metabolism has also emerged as a critical developmental regulator1-10, independent of its functions in energy production and growth. The mechanistic role of nutrient utilization in instructing cellular programmes to shape the in vivo developing mammalian embryo remains unknown. Here we reveal two spatially resolved, cell-type- and stage-specific waves of glucose metabolism during mammalian gastrulation by using single-cell-resolution quantitative imaging of developing mouse embryos, stem cell models and embryo-derived tissue explants. We identify that the first spatiotemporal wave of glucose metabolism occurs through the hexosamine biosynthetic pathway to drive fate acquisition in the epiblast, and the second wave uses glycolysis to guide mesoderm migration and lateral expansion. Furthermore, we demonstrate that glucose exerts its influence on these developmental processes through cellular signalling pathways, with distinct mechanisms connecting glucose with the ERK activity in each wave. Our findings underscore that-in synergy with genetic mechanisms and morphogenic gradients-compartmentalized cellular metabolism is integral in guiding cell fate and specialized functions during development. This study challenges the view of the generic and housekeeping nature of cellular metabolism, offering valuable insights into its roles in various developmental contexts.
    DOI:  https://doi.org/10.1038/s41586-024-08044-1
  8. Dev Cell. 2024 Oct 14. pii: S1534-5807(24)00545-8. [Epub ahead of print]
    Mesodermal fibronectin controls cell shape, polarity, and mechanotransduction in the second heart field during cardiac outflow tract development.
    Cecilia Arriagada, Evan Lin, Michael Schonning, Sophie Astrof.
      Failure in the elongation of the cardiac outflow tract (OFT) results in congenital heart disease due to the misalignment of the great arteries with the left and right ventricles. The OFT lengthens via the accretion of progenitors from the second heart field (SHF). SHF cells are exquisitely regionalized and organized into an epithelial-like layer, forming the dorsal pericardial wall (DPW). Tissue tension, cell polarity, and proliferation within the DPW are important for the addition of SHF-derived cells to the heart and OFT elongation. However, the genes controlling these processes are not completely characterized. Using conditional mutagenesis in the mouse, we show that fibronectin (FN1) synthesized by the mesoderm coordinates multiple cellular behaviors in the anterior DPW. FN1 is enriched in the anterior DPW and plays a role in OFT elongation by maintaining a balance between pro- and anti-adhesive cell-extracellular matrix (ECM) interactions and controlling DPW cell shape, polarity, cohesion, proliferation, and mechanotransduction.
    Keywords:  cardiac outflow tract; fibronectin; great arteries; heart development; second heart field; tenascin C
    DOI:  https://doi.org/10.1016/j.devcel.2024.09.017
  9. Dev Cell. 2024 Oct 14. pii: S1534-5807(24)00574-4. [Epub ahead of print]
    LINE1 and PRC2 control nucleolar organization and repression of the 8C state in human ESCs.
    Juan Zhang, Lamisa Ataei, Kirti Mittal, Liang Wu, Lauren Caldwell, Linh Huynh, Shahil Sarajideen, Kevin Tse, Marie-Michelle Simon, Md Abdul Mazid, David P Cook, Daniel Trcka, Tony Kwan, Michael M Hoffman, Jeffrey L Wrana, Miguel A Esteban, Miguel Ramalho-Santos.
      The mechanisms that ensure developmental progression in the early human embryo remain largely unknown. Here, we show that the family of long interspersed nuclear element 1 (LINE1) transposons prevents the reversion of naive human embryonic stem cells (hESCs) to 8-cell-like cells (8CLCs). LINE1 RNA contributes to maintenance of H3K27me3 levels, particularly at chromosome 19 (Chr19). Chr19 is enriched for key 8C regulators, H3K27me3, and genes derepressed upon LINE1 knockdown or PRC2 inhibition. Moreover, Chr19 is strongly associated with the nucleolus in hESCs but less in 8CLCs. Direct inhibition of PRC2 activity induces the 8C program and leads to a relocalization of Chr19 away from the nucleolus. LINE1 KD or PRC2 inhibition induces nucleolar stress, and disruption of nucleolar architecture is sufficient to de-repress the 8C program. These results indicate that LINE1 RNA and PRC2 maintain H3K27me3-mediated gene repression and 3D nuclear organization to prevent developmental reversion of hESCs.
    Keywords:  8-cell-like cells; H3K27me3; LINE1; chromosome 19; human embryonic stem cells; nuclear compartmentalization; nucleolus; polycomb repressive complex 2
    DOI:  https://doi.org/10.1016/j.devcel.2024.09.024
  10. Dev Cell. 2024 Oct 10. pii: S1534-5807(24)00546-X. [Epub ahead of print]
    RNA splicing controls organ-wide maturation of postnatal heart in mice.
    Zheng Li, Changchang Cao, Quanyi Zhao, Dandan Li, Yan Han, Mingzhi Zhang, Lin Mao, Bingying Zhou, Li Wang.
      Postnatal cardiac development requires the orchestrated maturation of diverse cellular components for which unifying control mechanisms are still lacking. Using full-length sequencing, we examined the transcriptomic landscape of the maturating mouse heart (E18.5-P28) at single-cell and transcript isoform resolution. We identified dynamically changing intercellular networks as a molecular basis of the maturing heart and alternative splicing (AS) as a common mechanism that distinguished developmental age. Manipulation of RNA-binding proteins (RBPs) remodeled the AS landscape, cardiac cell maturation, and intercellular communication through direct binding of splice targets, which were enriched for functions related to general, as well as cell-type-specific, maturation. Overexpression of an RBP nuclear cap-binding protein subunit 2 (NCBP2) in neonatal hearts repressed cardiac maturation. Together, our data suggest AS regulation by RBPs as an organ-level control mechanism in mammalian postnatal cardiac development and provide insight into the possibility of manipulating RBPs for therapeutic purposes.
    Keywords:  Ncbp2; RNA-binding protein; alternative splicing; cellular crosstalk; organ maturation; postnatal heart development
    DOI:  https://doi.org/10.1016/j.devcel.2024.09.018
  11. Dev Cell. 2024 Oct 08. pii: S1534-5807(24)00540-9. [Epub ahead of print]
    Zonated Wnt/β-catenin signal-activated cardiomyocytes at the atrioventricular canal promote coronary vessel formation in zebrafish.
    Ayano Chiba, Takuya Yamamoto, Hajime Fukui, Moe Fukumoto, Manabu Shirai, Hiroyuki Nakajima, Naoki Mochizuki.
      Cells functioning at a specific zone by clustering according to gene expression are recognized as zonated cells. Here, we demonstrate anatomical and functional zones in the zebrafish heart. The cardiomyocytes (CMs) at the atrioventricular canal between the atrium and ventricle could be grouped into three zones according to the localization of signal-activated CMs: Wnt/β-catenin signal+, Bmp signal+, and Tbx2b+ zones. Endocardial endothelial cells (ECs) changed their characteristics, penetrated the Wnt/β-catenin signal+ CM zone, and became coronary ECs covering the heart. Coronary vessel length was reduced when the Wnt/β-catenin signal+ CMs were depleted. Collectively, we demonstrate the importance of anatomical and functional zonation of CMs in the zebrafish heart.
    Keywords:  Wnt/β-catenin signaling; angiogenesis; arterialization; atrioventricular canal; coronary vessels; heartbeat; zonation
    DOI:  https://doi.org/10.1016/j.devcel.2024.09.012
  12. Autophagy. 2024 Oct 14. 1-3
    Mitophagy as a guardian against cellular aging.
    Tetsushi Kataura, Niall Wilson, Gailing Ma, Viktor I Korolchuk.
      Mitophagy, the selective autophagic clearance of damaged mitochondria, is considered vital for maintaining mitochondrial quality and cellular homeostasis; however, its molecular mechanisms, particularly under basal conditions, and its role in cellular physiology remain poorly characterized. We recently demonstrated that basal mitophagy is a key feature of primary human cells and is downregulated by immortalization, suggesting its dependence on the primary cell state. Mechanistically, we demonstrated that the PINK1-PRKN-SQSTM1 pathway regulates basal mitophagy, with SQSTM1 sensing superoxide-enriched mitochondria through its redox-sensitive cysteine residues, which mediate SQSTM1 oligomerization and mitophagy activation. We developed STOCK1N-57534, a small molecule that targets and promotes this SQSTM1 activation mechanism. Treatment with STOCK1N-57534 reactivates mitophagy downregulated in senescent and naturally aged donor-derived primary cells, improving cellular senescence(-like) phenotypes. Our findings highlight that basal mitophagy is protective against cellular senescence and aging, positioning its pharmacological reactivation as a promising anti-aging strategy.Abbreviation: IR: ionizing radiation; ROS: reactive oxygen species; SARs: selective autophagy receptors.
    Keywords:  Aging; SQSTM1/p62; autophagy; mitochondria; mitophagy; senescence
    DOI:  https://doi.org/10.1080/15548627.2024.2414461
  13. J Mol Cell Biol. 2024 Oct 11. pii: mjae044. [Epub ahead of print]
    HDAC7 promotes cardiomyocyte proliferation by suppressing Myocyte Enhancer Factor 2.
    Jihyun Jang, Mette Bentsen, Jin Bu, Ling Chen, Alexandre Rosa Campos, Mario Looso, Deqiang Li.
      Postnatal mammalian cardiomyocytes (CMs) rapidly lose proliferative capacity and exit the cell cycle and undergo further differentiation and maturation. Cell cycle activation has been a major strategy to stimulate postnatal CM proliferation, albeit achieving modest effects. One impediment is that postnatal CMs may need to undergo dedifferentiation before proliferation, if not simultaneously. Here, we report that overexpression of Hdac7 in neonatal mouse CMs results in significant CM dedifferentiation and proliferation. Mechanistically, we show that HDAC7-mediated CM proliferation is contingent on dedifferentiation, which is accomplished through suppressing MEF2. Hdac7 overexpression in CM shifts the chromatin state from binding MEF2, which favors the differentiation transcriptional program to AP-1, which favors the proliferative transcriptional program. Further, we found that HDAC7 interacts with minichromosome maintenance complex (MCM) components to initiate cell cycle progression. Our findings reveal that HDAC7 promotes CM proliferation by its dual action on CM dedifferentiation and proliferation, uncovering a potential new strategy for heart regeneration/repair.
    Keywords:  HDAC7; cardiomyocyte; dedifferentiation; proliferation
    DOI:  https://doi.org/10.1093/jmcb/mjae044
  14. Curr Biol. 2024 Oct 15. pii: S0960-9822(24)01150-3. [Epub ahead of print]
    Kinesin-5/Cut7 C-terminal tail phosphorylation is essential for microtubule sliding force and bipolar mitotic spindle assembly.
    Michele H Jones, Zachary R Gergely, Daniel Steckhahn, Bojun Zhou, Meredith D Betterton.
      Kinesin-5 motors play an essential role during mitotic spindle assembly in many organisms1,2,3,4,5,6,7,8,9,10,11: they crosslink antiparallel spindle microtubules, step toward plus ends, and slide the microtubules apart.12,13,14,15,16,17 This activity separates the spindle poles and chromosomes. Kinesin-5s are not only plus-end-directed but can walk or be carried toward MT minus ends,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 where they show enhanced localization.3,5,7,27,29,32 The kinesin-5 C-terminal tail interacts with and regulates the motor, affecting structure, motility, and sliding force of purified kinesin-535,36,37 along with motility and spindle assembly in cells.27,38,39 The tail contains phosphorylation sites, particularly in the conserved BimC box.6,7,40,41,42,43,44 Nine mitotic tail phosphorylation sites were identified in the kinesin-5 motor of the fission yeast Schizosaccharomyces pombe,45,46,47,48 suggesting that multi-site phosphorylation may regulate kinesin-5s. Here, we show that mutating all nine sites to either alanine or glutamate causes temperature-sensitive lethality due to a failure of bipolar spindle assembly. We characterize kinesin-5 localization and sliding force in the spindle based on Cut7-dependent microtubule minus-end protrusions in cells lacking kinesin-14 motors.39,49,50,51,52 Imaging and computational modeling show that Cut7p simultaneously moves toward the minus ends of protrusion MTs and the plus ends of spindle midzone MTs. Phosphorylation mutants show dramatic decreases in protrusions and sliding force. Comparison to a model of force to create protrusions suggests that tail truncation and phosphorylation mutants decrease Cut7p sliding force similarly to tail-truncated human Eg5.36 Our results show that C-terminal tail phosphorylation is required for kinesin-5/Cut7 sliding force and bipolar spindle assembly in fission yeast.
    Keywords:  fission yeast; kinesin-5; microtubule; microtubule sliding; mitosis; mitotic spindle; modeling; motor protein; phosphoregulation; spindle assembly
    DOI:  https://doi.org/10.1016/j.cub.2024.08.035
  15. Proc Natl Acad Sci U S A. 2024 Oct 22. 121(43): e2405463121
    Single-cell resolution of intestinal regeneration in pythons without crypts illuminates conserved vertebrate regenerative mechanisms.
    Aundrea K Westfall, Siddharth S Gopalan, Jarren C Kay, Trevor S Tippetts, Margaret B Cervantes, Kimberly Lackey, Saiful M Chowdhury, Mark W Pellegrino, Todd A Castoe.
      Canonical models of intestinal regeneration emphasize the critical role of the crypt stem cell niche to generate enterocytes that migrate to villus ends. Burmese pythons possess extreme intestinal regenerative capacity yet lack crypts, thus providing opportunities to identify noncanonical but potentially conserved mechanisms that expand our understanding of regenerative capacity in vertebrates, including humans. Here, we leverage single-nucleus RNA sequencing of fasted and postprandial python small intestine to identify the signaling pathways and cell-cell interactions underlying the python's regenerative response. We find that python intestinal regeneration entails the activation of multiple conserved mechanisms of growth and stress response, including core lipid metabolism pathways and the unfolded protein response in intestinal enterocytes. Our single-cell resolution highlights extensive heterogeneity in mesenchymal cell population signaling and intercellular communication that directs major tissue restructuring and the shift out of a dormant fasted state by activating both embryonic developmental and wound healing pathways. We also identify distinct roles of BEST4+ enterocytes in coordinating key regenerative transitions via NOTCH signaling. Python intestinal regeneration shares key signaling features and molecules with mammalian gastric bypass, indicating that conserved regenerative programs are common to both. Our findings provide different insights into cooperative and conserved regenerative programs and intercellular interactions in vertebrates independent of crypts which have been otherwise obscured in model species where temporal phases of generative growth are limited to embryonic development or recovery from injury.
    Keywords:  BEST4+ cells; NOTCH signaling; RYGB; lipid metabolism; stress response
    DOI:  https://doi.org/10.1073/pnas.2405463121
  16. Nat Struct Mol Biol. 2024 Oct 18.
    Mechanism of polyadenylation-independent RNA polymerase II termination.
    Srinivasan Rengachari, Thomas Hainthaler, Christiane Oberthuer, Michael Lidschreiber, Patrick Cramer.
      The mechanisms underlying the initiation and elongation of RNA polymerase II (Pol II) transcription are well-studied, whereas termination remains poorly understood. Here we analyze the mechanism of polyadenylation-independent Pol II termination mediated by the yeast Sen1 helicase. Cryo-electron microscopy structures of two pretermination intermediates show that Sen1 binds to Pol II and uses its adenosine triphosphatase activity to pull on exiting RNA in the 5' direction. This is predicted to push Pol II forward, induce an unstable hypertranslocated state and destabilize the transcription bubble, thereby facilitating termination. This mechanism of transcription termination may be widely used because it is conceptually conserved in the bacterial transcription system.
    DOI:  https://doi.org/10.1038/s41594-024-01409-0
  17. Nat Commun. 2024 Oct 15. 15(1): 8887
    The subcortical maternal complex modulates the cell cycle during early mammalian embryogenesis via 14-3-3.
    Zhuo Han, Rui Wang, Pengliang Chi, Zihan Zhang, Ling Min, Haizhan Jiao, Guojin Ou, Dan Zhou, Dandan Qin, Chengpeng Xu, Zheng Gao, Qianqian Qi, Jialu Li, Yuechao Lu, Xiang Wang, Jing Chen, Xingjiang Yu, Hongli Hu, Lei Li, Dong Deng.
      The subcortical maternal complex (SCMC) is essential for safeguarding female fertility in mammals. Assembled in oocytes, the SCMC maintains the cleavage of early embryos, but the underlying mechanism remains unclear. Here, we report that 14-3-3, a multifunctional protein, is a component of the SCMC. By resolving the structure of the 14-3-3-containing SCMC, we discover that phosphorylation of TLE6 contributes to the recruitment of 14-3-3. Mechanistically, during maternal-to-embryo transition, the SCMC stabilizes 14-3-3 protein and contributes to the proper control of CDC25B, thus ensuring the activation of the maturation-promoting factor and mitotic entry in mouse zygotes. Notably, the SCMC establishes a conserved molecular link with 14-3-3 and CDC25B in human oocytes/embryos. This study discloses the molecular mechanism through which the SCMC regulates the cell cycle in early embryos and elucidates the function of the SCMC in mammalian early embryogenesis.
    DOI:  https://doi.org/10.1038/s41467-024-53277-3
  18. bioRxiv. 2024 Oct 11. pii: 2024.10.10.617645. [Epub ahead of print]
    cGAS deficient mice display premature aging associated with de-repression of LINE1 elements and inflammation.
    John C Martinez, Francesco Morandini, Lucinda Fitzgibbons, Natasha Sieczkiewicz, Sung Jae Bae, Michael E Meadow, Eric Hillpot, Joseph Cutting, Victoria Paige, Seyed Ali Biashad, Matthew Simon, John Sedivy, Andrei Seluanov, Vera Gorbunova.
      Aging-associated inflammation, or 'inflammaging" is a driver of multiple age-associated diseases. Cyclic GMP-AMP Synthase (cGAS) is a cytosolic DNA sensor that functions to activate interferon response upon detecting viral DNA in the cytoplasm. cGAS contributes to inflammaging by responding to endogenous signals such as damaged DNA or LINE1 (L1) cDNA which forms in aged cells. While cGAS knockout mice are viable their aging has not been examined. Unexpectedly, we found that cGAS knockout mice exhibit accelerated aging phenotype associated with induction of inflammation. Transcription of L1 elements was increased in both cGAS knockout mice and in cGAS siRNA knockdown cells associated with high levels of cytoplasmic L1 DNA and expression of ORF1 protein. Cells from cGAS knockout mice showed increased chromatin accessibility and decreased DNA methylation on L1 transposons. Stimulated emission depletion microscopy (STED) showed that cGAS forms nuclear condensates that co-localize with H3K9me3 heterochromatin marks, and H3K9me3 pattern is disrupted in cGAS knockout cells. Taken together these results suggest a previously undescribed role for cGAS in maintaining heterochromatin on transposable elements. We propose that loss of cGAS leads to loss of chromatin organization, de-repression of transposable elements and induction of inflammation resulting in accelerated aging.
    DOI:  https://doi.org/10.1101/2024.10.10.617645
  19. Cell. 2024 Oct 12. pii: S0092-8674(24)01092-4. [Epub ahead of print]
    Multiscale drug screening for cardiac fibrosis identifies MD2 as a therapeutic target.
    Hao Zhang, Phung N Thai, Rabindra V Shivnaraine, Lu Ren, Xuekun Wu, Dirk H Siepe, Yu Liu, Chengyi Tu, Hye Sook Shin, Arianne Caudal, Souhrid Mukherjee, Jeremy Leitz, Wilson Tan Lek Wen, Wenqiang Liu, Wenjuan Zhu, Nipavan Chiamvimonvat, Joseph C Wu.
      Cardiac fibrosis impairs cardiac function, but no effective clinical therapies exist. To address this unmet need, we employed a high-throughput screening for antifibrotic compounds using human induced pluripotent stem cell (iPSC)-derived cardiac fibroblasts (CFs). Counter-screening of the initial candidates using iPSC-derived cardiomyocytes and iPSC-derived endothelial cells excluded hits with cardiotoxicity. This screening process identified artesunate as the lead compound. Following profibrotic stimuli, artesunate inhibited proliferation, migration, and contraction in human primary CFs, reduced collagen deposition, and improved contractile function in 3D-engineered heart tissues. Artesunate also attenuated cardiac fibrosis and improved cardiac function in heart failure mouse models. Mechanistically, artesunate targeted myeloid differentiation factor 2 (MD2) and inhibited MD2/Toll-like receptor 4 (TLR4) signaling pathway, alleviating fibrotic gene expression in CFs. Our study leverages multiscale drug screening that integrates a human iPSC platform, tissue engineering, animal models, in silico simulations, and multiomics to identify MD2 as a therapeutic target for cardiac fibrosis.
    Keywords:  artesunate; cardiac fibrosis; cardiovascular; drug screening; induced pluripotent stem cells
    DOI:  https://doi.org/10.1016/j.cell.2024.09.034
  20. Aging Biol. 2023 ;pii: 20230005. [Epub ahead of print]1(1):
    Analysis of Somatic Mutations in Senescent Cells Using Single-Cell Whole-Genome Sequencing.
    Lei Zhang, Marco De Cecco, Moonsook Lee, Xiaoxiao Hao, Alexander Y Maslov, Cristina Montagna, Judith Campisi, Xiao Dong, John M Sedivy, Jan Vijg.
      Somatic mutations accumulate in multiple organs and tissues during aging and are a known cause of cancer. Cellular senescence is a possible cause of functional decline in aging, yet also acts as an anticancer mechanism in vivo. Here, we compared somatic mutation burden between early passage and deeply senescent human fibroblasts using single-cell whole-genome sequencing. The results show that single-nucleotide variants (SNVs) and small insertions and deletions (INDELs) are increased in senescent cells by about twofold but have the same mutational signature as early passage cells. The increase in SNVs and INDELs can be explained by increased replication errors due to the increased number of cell divisions senescent cells are likely to have undergone. By contrast, a stark increase of aneuploidies was observed in deeply senescent cells, with about half of all senescent cells affected but none of the early passage cells analyzed. These results indicate that large chromosomal events rather than small base substitutions or insertions and deletions could be mechanistically linked to cellular senescence.
    DOI:  https://doi.org/10.59368/agingbio.20230005
  21. Mol Cell Proteomics. 2024 Oct 14. pii: S1535-9476(24)00152-X. [Epub ahead of print] 100862
    Integrative omics reveals the metabolic patterns during oocyte growth.
    Xiang Zhang, Juan Ge, Yue Wang, Minjian Chen, Xuejiang Guo, Shuai Zhu, Hui Wang, Qiang Wang.
      Well-controlled metabolism is associated with high quality of oocytes and optimal development of a healthy embryo. However, the metabolic framework that controls mammalian oocyte growth remains unknown. In the present study, we comprehensively depict the temporal metabolic dynamics of mouse oocytes during in vivo growth through the integrated analysis of metabolomics and proteomics. A number of novel metabolic features are discovered during this process. Of note, glycolysis is enhanced and oxidative phosphorylation capacity is reduced in the growing oocytes, presenting a Warburg-like metabolic program. For the nucleotide biosynthesis, the salvage pathway is markedly activated during oocyte growth, whereas the de novo pathway is evidently suppressed. Fatty acid synthesis and channeling into phosphoinositides are specifically elevated in oocytes accompanying primordial follicle activation; nevertheless, fatty acid oxidation is reduced in these oocytes simultaneously. Our data establish the metabolic landscape during in vivo oocyte growth and serve as a broad resource for probing mammalian oocyte metabolism.
    Keywords:  Metabolism; follicle; oocyte; proteomics; reproduction
    DOI:  https://doi.org/10.1016/j.mcpro.2024.100862
  22. Nature. 2024 Oct 16.
    Quantifying constraint in the human mitochondrial genome.
    Nicole J Lake, Kaiyue Ma, Wei Liu, Stephanie L Battle, Kristen M Laricchia, Grace Tiao, Daniela Puiu, Kenneth K Ng, Justin Cohen, Alison G Compton, Shannon Cowie, John Christodoulou, David R Thorburn, Hongyu Zhao, Dan E Arking, Shamil R Sunyaev, Monkol Lek.
      Mitochondrial DNA (mtDNA) has an important yet often overlooked role in health and disease. Constraint models quantify the removal of deleterious variation from the population by selection and represent powerful tools for identifying genetic variation that underlies human phenotypes1-4. However, nuclear constraint models are not applicable to mtDNA, owing to its distinct features. Here we describe the development of a mitochondrial genome constraint model and its application to the Genome Aggregation Database (gnomAD), a large-scale population dataset that reports mtDNA variation across 56,434 human participants5. Specifically, we analyse constraint by comparing the observed variation in gnomAD to that expected under neutrality, which was calculated using a mtDNA mutational model and observed maximum heteroplasmy-level data. Our results highlight strong depletion of expected variation, which suggests that many deleterious mtDNA variants remain undetected. To aid their discovery, we compute constraint metrics for every mitochondrial protein, tRNA and rRNA gene, which revealed a range of intolerance to variation. We further characterize the most constrained regions within genes through regional constraint and identify the most constrained sites within the entire mitochondrial genome through local constraint, which showed enrichment of pathogenic variation. Constraint also clustered in three-dimensional structures, which provided insight into functionally important domains and their disease relevance. Notably, we identify constraint at often overlooked sites, including in rRNA and noncoding regions. Last, we demonstrate that these metrics can improve the discovery of deleterious variation that underlies rare and common phenotypes.
    DOI:  https://doi.org/10.1038/s41586-024-08048-x
  23. Cell. 2024 Oct 03. pii: S0092-8674(24)01087-0. [Epub ahead of print]
    Structural basis of respiratory complex adaptation to cold temperatures.
    Young-Cheul Shin, Pedro Latorre-Muro, Amina Djurabekova, Oleksii Zdorevskyi, Christopher F Bennett, Nils Burger, Kangkang Song, Chen Xu, Joao A Paulo, Steven P Gygi, Vivek Sharma, Maofu Liao, Pere Puigserver.
      In response to cold, mammals activate brown fat for respiratory-dependent thermogenesis reliant on the electron transport chain. Yet, the structural basis of respiratory complex adaptation upon cold exposure remains elusive. Herein, we combined thermoregulatory physiology and cryoelectron microscopy (cryo-EM) to study endogenous respiratory supercomplexes from mice exposed to different temperatures. A cold-induced conformation of CI:III2 (termed type 2) supercomplex was identified with a ∼25° rotation of CIII2 around its inter-dimer axis, shortening inter-complex Q exchange space, and exhibiting catalytic states that favor electron transfer. Large-scale supercomplex simulations in mitochondrial membranes reveal how lipid-protein arrangements stabilize type 2 complexes to enhance catalytic activity. Together, our cryo-EM studies, multiscale simulations, and biochemical analyses unveil the thermoregulatory mechanisms and dynamics of increased respiratory capacity in brown fat at the structural and energetic level.
    Keywords:  CIII(2) rotation; brown adipose tissue; cellular adaptation; electron transport chain; membrane lipid remodeling; respiratory complexes
    DOI:  https://doi.org/10.1016/j.cell.2024.09.029
  24. PLoS Comput Biol. 2024 Oct 14. 20(10): e1012465
    Emergent order in epithelial sheets by interplay of cell divisions and cell fate regulation.
    Philip Greulich.
      The fate choices of stem cells between self-renewal and differentiation are often tightly regulated by juxtacrine (cell-cell contact) signalling. Here, we assess how the interplay between cell division, cell fate choices, and juxtacrine signalling can affect the macroscopic ordering of cell types in self-renewing epithelial sheets, by studying a simple spatial cell fate model with cells being arranged on a 2D lattice. We show in this model that if cells commit to their fate directly upon cell division, macroscopic patches of cells of the same type emerge, if at least a small proportion of divisions are symmetric, except if signalling interactions are laterally inhibiting. In contrast, if cells are first 'licensed' to differentiate, yet retaining the possibility to return to their naive state, macroscopic order only emerges if the signalling strength exceeds a critical threshold: if then the signalling interactions are laterally inducing, macroscopic patches emerge as well. Lateral inhibition, on the other hand, can in that case generate periodic patterns of alternating cell types (checkerboard pattern), yet only if the proportion of symmetric divisions is sufficiently low. These results can be understood theoretically by an analogy to phase transitions in spin systems known from statistical physics.
    DOI:  https://doi.org/10.1371/journal.pcbi.1012465
  25. Dev Cell. 2024 Oct 12. pii: S1534-5807(24)00572-0. [Epub ahead of print]
    Ataxin-2 polyglutamine expansions aberrantly sequester TDP-43 ribonucleoprotein condensates disrupting mRNA transport and local translation in neurons.
    Denethi Wijegunawardana, Asima Nayak, Sonali S Vishal, Neha Venkatesh, Pallavi P Gopal.
      Altered RNA metabolism and misregulation of transactive response DNA-binding protein of 43 kDa (TDP-43), an essential RNA-binding protein (RBP), define amyotrophic lateral sclerosis (ALS). Intermediate-length polyglutamine (polyQ) expansions of Ataxin-2, a like-Sm (LSm) RBP, are associated with increased risk for ALS, but the underlying biological mechanisms remain unknown. Here, we studied the spatiotemporal dynamics and mRNA regulatory functions of TDP-43 and Ataxin-2 ribonucleoprotein (RNP) condensates in rodent (rat) primary cortical neurons and mouse motor neuron axons in vivo. We report that Ataxin-2 polyQ expansions aberrantly sequester TDP-43 within RNP condensates and disrupt both its motility along the axon and liquid-like properties. We provide evidence that Ataxin-2 governs motility and translation of neuronal RNP condensates and that Ataxin-2 polyQ expansions fundamentally perturb spatial localization of mRNA and suppress local translation. Overall, our results support a model in which Ataxin-2 polyQ expansions disrupt stability, localization, and/or translation of critical axonal and cytoskeletal mRNAs, particularly important for motor neuron integrity.
    Keywords:  Ataxin-2; TDP-43; amyotrophic lateral sclerosis; axonal transport; liquid-liquid phase separation; local translation; mRNA localization; neuronal cell biology; neuronal transport granules; ribonucleoprotein condensates
    DOI:  https://doi.org/10.1016/j.devcel.2024.09.023
  26. Cell. 2024 Oct 03. pii: S0092-8674(24)01068-7. [Epub ahead of print]
    Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex.
    Owen T Tuck, Benjamin A Adler, Emily G Armbruster, Arushi Lahiri, Jason J Hu, Julia Zhou, Joe Pogliano, Jennifer A Doudna.
      Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman is a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact double-stranded DNA (dsDNA). When the HamAB complex detects DNA damage, HamB helicase activity activates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating "phantom" cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and enzymes from eukaryotes and archaea suggest deep functional symmetries with other important helicases across domains of life.
    Keywords:  cryo-EM; genome integrity; helicase; immunity; phage defense
    DOI:  https://doi.org/10.1016/j.cell.2024.09.020
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