bims-ginsta Biomed News
on Genome instability
Issue of 2024–02–18
27 papers selected by
Jinrong Hu, National University of Singapore
  1. A conserved CENP-E region mediates BubR1-independent recruitment to the outer corona at mitotic onset.
  2. Local monomer levels and established filaments potentiate non-muscle myosin 2 assembly.
  3. Nuclear morphology is shaped by loop-extrusion programs.
  4. Specialized replication mechanisms maintain genome stability at human centromeres.
  5. Tbx5 maintains atrial identity in post-natal cardiomyocytes by regulating an atrial-specific enhancer network.
  6. Polycomb repression of Hox genes involves spatial feedback but not domain compaction or phase transition.
  7. E-cadherin biomaterials reprogram collective cell migration and cell cycling by forcing homeostatic conditions.
  8. Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA.
  9. Vinculin is essential for sustaining normal levels of endogenous forces at cell-cell contacts.
  10. Lineage motifs as developmental modules for control of cell type proportions.
  11. Leveraging chromatin state transitions for the identification of regulatory networks orchestrating heart regeneration.
  12. Unraveling the interplay between PKA inhibition and Cdk1 activation during oocyte meiotic maturation.
  13. Applying mechanical forces on Drosophila tissues in vivo using the StretchCo, a 3D-printable device.
  14. Nuclear mechanotransduction on skin stem cell fate regulation.
  15. Proline restores mitochondrial function and reverses aging hallmarks in senescent cells.
  16. NELF coordinates Pol II transcription termination and DNA replication initiation.
  17. Chp1 is a dedicated chaperone at the ribosome that safeguards eEF1A biogenesis.
  18. Cloning and expansion of repetitive DNA sequences.
  19. ATR blocks telomerase from converting DNA breaks into telomeres.
  20. Three dimensional fibrotic extracellular matrix directs microenvironment fiber remodeling by fibroblasts.
  21. Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition.
  22. In situ structure of actin remodeling during glucose-stimulated insulin secretion using cryo-electron tomography.
  23. Generation of aneuploid cells and assessment of their ability to survive in presence of chemotherapeutic agents.
  24. Genetic determinants of micronucleus formation in vivo.
  25. Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility.
  26. Biphasic regulation of epigenetic state by matrix stiffness during cell reprogramming.
  27. A single-cell time-lapse of mouse prenatal development from gastrula to birth.
  1. Curr Biol. 2024 Feb 08. pii: S0960-9822(24)00076-9. [Epub ahead of print]
    A conserved CENP-E region mediates BubR1-independent recruitment to the outer corona at mitotic onset.
    Jeraldine Weber, Thibault Legal, Alicia Perez Lezcano, Agata Gluszek-Kustusz, Calum Paterson, Susana Eibes, Marin Barisic, Owen R Davies, Julie P I Welburn.
      The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores.1,2 The low-density structure of the corona forms through the expansion of unattached kinetochores. It comprises the RZZ complex, the dynein adaptor Spindly, the plus-end directed microtubule motor centromere protein E (CENP-E), and the Mad1/Mad2 spindle-assembly checkpoint proteins.3,4,5,6,7,8,9,10 CENP-E specifically associates with unattached kinetochores to facilitate chromosome congression,11,12,13,14,15,16 interacting with BubR1 at the kinetochore through its C-terminal region (2091-2358).17,18,19,20,21 We recently showed that CENP-E recruitment to BubR1 at the kinetochores is both rapid and essential for correct chromosome alignment. However, CENP-E is also recruited to the outer corona by a second, slower pathway that is currently undefined.19 Here, we show that BubR1-independent localization of CENP-E is mediated by a conserved loop that is essential for outer-corona targeting. We provide a structural model of the entire CENP-E kinetochore-targeting domain combining X-ray crystallography and Alphafold2. We reveal that maximal recruitment of CENP-E to unattached kinetochores critically depends on BubR1 and the outer corona, including dynein. Ectopic expression of the CENP-E C-terminal domain recruits the RZZ complex, Mad1, and Spindly, and prevents kinetochore biorientation in cells. We propose that BubR1-recruited CENP-E, in addition to its essential role in chromosome alignment to the metaphase plate, contributes to the recruitment of outer corona proteins through interactions with the CENP-E corona-targeting domain to facilitate the rapid capture of microtubules for efficient chromosome alignment and mitotic progression.
    Keywords:  CENP-E motor; centromere; dynein; kinetochore; microtubule; mitosis; motor; outer corona; prometaphase; spindle assembly checkpoint
    DOI:  https://doi.org/10.1016/j.cub.2024.01.042
  2. J Cell Biol. 2024 Apr 01. pii: e202305023. [Epub ahead of print]223(4):
    Local monomer levels and established filaments potentiate non-muscle myosin 2 assembly.
    Melissa A Quintanilla, Hiral Patel, Huini Wu, Kem A Sochacki, Shreya Chandrasekar, Matthew Akamatsu, Jeremy D Rotty, Farida Korobova, James E Bear, Justin W Taraska, Patrick W Oakes, Jordan R Beach.
      The ability to dynamically assemble contractile networks is required throughout cell physiology, yet direct biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here, we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the static actin architecture plays a less clear role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin-driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes filament stacks prior to partitioning into clusters that feed higher-order networks. Together, these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.
    DOI:  https://doi.org/10.1083/jcb.202305023
  3. Nature. 2024 Feb 14.
    Nuclear morphology is shaped by loop-extrusion programs.
    Indumathi Patta, Maryam Zand, Lindsay Lee, Shreya Mishra, Alexandra Bortnick, Hanbin Lu, Arpita Prusty, Sara McArdle, Zbigniew Mikulski, Huan-You Wang, Christine S Cheng, Kathleen M Fisch, Ming Hu, Cornelis Murre.
      It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces1-3. However, how polymorphonuclear structures are assembled remains unknown4. Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin5-leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted in enrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments.
    DOI:  https://doi.org/10.1038/s41586-024-07086-9
  4. Mol Cell. 2024 Feb 14. pii: S1097-2765(24)00054-6. [Epub ahead of print]
    Specialized replication mechanisms maintain genome stability at human centromeres.
    Andrea Scelfo, Annapaola Angrisani, Marco Grillo, Bethany M Barnes, Francesc Muyas, Carolin M Sauer, Chin Wei Brian Leung, Marie Dumont, Marine Grison, David Mazaud, Mickaël Garnier, Laetitia Guintini, Louisa Nelson, Fumiko Esashi, Isidro Cortés-Ciriano, Stephen S Taylor, Jérôme Déjardin, Therese Wilhelm, Daniele Fachinetti.
      The high incidence of whole-arm chromosome aneuploidy and translocations in tumors suggests instability of centromeres, unique loci built on repetitive sequences and essential for chromosome separation. The causes behind this fragility and the mechanisms preserving centromere integrity remain elusive. We show that replication stress, hallmark of pre-cancerous lesions, promotes centromeric breakage in mitosis, due to spindle forces and endonuclease activities. Mechanistically, we unveil unique dynamics of the centromeric replisome distinct from the rest of the genome. Locus-specific proteomics identifies specialized DNA replication and repair proteins at centromeres, highlighting them as difficult-to-replicate regions. The translesion synthesis pathway, along with other factors, acts to sustain centromere replication and integrity. Prolonged stress causes centromeric alterations like ruptures and translocations, as observed in ovarian cancer models experiencing replication stress. This study provides unprecedented insights into centromere replication and integrity, proposing mechanistic insights into the origins of centromere alterations leading to abnormal cancerous karyotypes.
    Keywords:  DNA damage; DNA replication; cancer; centromere; genome instability; mitosis; proteomics; recombination; replication stress
    DOI:  https://doi.org/10.1016/j.molcel.2024.01.018
  5. Nat Cardiovasc Res. 2023 Oct;2(10): 881-898
    Tbx5 maintains atrial identity in post-natal cardiomyocytes by regulating an atrial-specific enhancer network.
    Mason E Sweat, Yangpo Cao, Xiaoran Zhang, Ozanna Burnicka-Turek, Carlos Perez-Cervantes, Kulandai Arulsamy, Fujian Lu, Erin M Keating, Brynn N Akerberg, Qing Ma, Hiroko Wakimoto, Joshua M Gorham, Lauren D Hill, Mi Kyoung Song, Michael A Trembley, Peizhe Wang, Matteo Gianeselli, Maksymilian Prondzynski, Raul H Bortolin, Vassilios J Bezzerides, Kaifu Chen, Jonathan G Seidman, Christine E Seidman, Ivan P Moskowitz, William T Pu.
      Understanding how the atrial and ventricular heart chambers maintain distinct identities is a prerequisite for treating chamber-specific diseases. Here, we selectively knocked out (KO) the transcription factor Tbx5 in the atrial working myocardium to evaluate its requirement for atrial identity. Atrial Tbx5 inactivation downregulated atrial cardiomyocyte (aCM) selective gene expression. Using concurrent single nucleus transcriptome and open chromatin profiling, genomic accessibility differences were identified between control and Tbx5 KO aCMs, revealing that 69% of the control-enriched ATAC regions were bound by TBX5. Genes associated with these regions were downregulated in KO aCMs, suggesting they function as TBX5-dependent enhancers. Comparing enhancer chromatin looping using H3K27ac HiChIP identified 510 chromatin loops sensitive to TBX5 dosage, and 74.8% of control-enriched loops contained anchors in control-enriched ATAC regions. Together, these data demonstrate TBX5 maintains the atrial gene expression program by binding to and preserving the tissue-specific chromatin architecture of atrial enhancers.
    DOI:  https://doi.org/10.1038/s44161-023-00334-7
  6. Nat Genet. 2024 Feb 15.
    Polycomb repression of Hox genes involves spatial feedback but not domain compaction or phase transition.
    Sedona Eve Murphy, Alistair Nicol Boettiger.
      Polycomb group proteins have a critical role in silencing transcription during development. It is commonly proposed that Polycomb-dependent changes in genome folding, which compact chromatin, contribute directly to repression by blocking the binding of activating complexes. Recently, it has also been argued that liquid-liquid demixing of Polycomb proteins facilitates this compaction and repression by phase-separating target genes into a membraneless compartment. To test these models, we used Optical Reconstruction of Chromatin Architecture to trace the Hoxa gene cluster, a canonical Polycomb target, in thousands of single cells. Across multiple cell types, we find that Polycomb-bound chromatin frequently explores decompact states and partial mixing with neighboring chromatin, while remaining uniformly repressed, challenging the repression-by-compaction or phase-separation models. Using polymer simulations, we show that these observed flexible ensembles can be explained by 'spatial feedback'-transient contacts that contribute to the propagation of the epigenetic state (epigenetic memory), without inducing a globular organization.
    DOI:  https://doi.org/10.1038/s41588-024-01661-6
  7. Cell Rep. 2024 Feb 13. pii: S2211-1247(24)00071-8. [Epub ahead of print]43(2): 113743
    E-cadherin biomaterials reprogram collective cell migration and cell cycling by forcing homeostatic conditions.
    Kevin Suh, Youn Kyoung Cho, Isaac B Breinyn, Daniel J Cohen.
      Cells attach to the world through either cell-extracellular matrix adhesion or cell-cell adhesion, and traditional biomaterials imitate the matrix for integrin-based adhesion. However, materials incorporating cadherin proteins that mimic cell-cell adhesion offer an alternative to program cell behavior and integrate into living tissues. We investigated how cadherin substrates affect collective cell migration and cell cycling in epithelia. Our approach involved biomaterials with matrix proteins on one-half and E-cadherin proteins on the other, forming a "Janus" interface across which we grew a single sheet of cells. Tissue regions over the matrix side exhibited normal collective dynamics, but an abrupt behavior shift occurred across the Janus boundary onto the E-cadherin side, where cells attached to the substrate via E-cadherin adhesions, resulting in stalled migration and slowing of the cell cycle. E-cadherin surfaces disrupted long-range mechanical coordination and nearly doubled the length of the G0/G1 phase of the cell cycle, linked to the lack of integrin focal adhesions on the E-cadherin surface.
    Keywords:  CP: Cell biology; E-cadherin; biomaterials; cell adhesion; cell cycle; collective migration
    DOI:  https://doi.org/10.1016/j.celrep.2024.113743
  8. Nat Struct Mol Biol. 2024 Feb 12.
    Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA.
    R Stefan Isaac, Thomas W Tullius, Katja G Hansen, Danilo Dubocanin, Mary Couvillion, Andrew B Stergachis, L Stirling Churchman.
      Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet because mitochondria lack histones or other machinery important for nuclear genome compaction, it remains unresolved how mtDNA is packaged into individual nucleoids. In this study, we used long-read single-molecule accessibility mapping to measure the compaction of individual full-length mtDNA molecules at near single-nucleotide resolution. We found that, unlike the nuclear genome, human mtDNA largely undergoes all-or-none global compaction, with most nucleoids existing in an inaccessible, inactive state. Highly accessible mitochondrial nucleoids are co-occupied by transcription and replication components and selectively form a triple-stranded displacement loop structure. In addition, we showed that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, acting consistently with a nucleation-and-spreading mechanism to coat and compact mitochondrial nucleoids. Together, these findings reveal the primary architecture of mtDNA packaging and regulation in human cells.
    DOI:  https://doi.org/10.1038/s41594-024-01225-6
  9. Biophys J. 2023 Dec 05. pii: S0006-3495(23)00665-3. [Epub ahead of print]122(23): 4518-4527
    Vinculin is essential for sustaining normal levels of endogenous forces at cell-cell contacts.
    Mazen Mezher, Sandeep Dumbali, Ian Fenn, Carter Lamb, Conrad Miller, Saika Sharmin, Jolene I Cabe, Vidal Bejar-Padilla, Daniel Conway, Venkat Maruthamuthu.
      Transmission of cell-generated (i.e., endogenous) tension at cell-cell contacts is crucial for tissue shape changes during morphogenesis and adult tissue repair in tissues such as epithelia. E-cadherin-based adhesions at cell-cell contacts are the primary means by which endogenous tension is transmitted between cells. The E-cadherin-β-catenin-α-catenin complex mechanically couples to the actin cytoskeleton (and thereby the cell's contractile machinery) both directly and indirectly. However, the key adhesion constituents required for substantial endogenous force transmission at these adhesions in cell-cell contacts are unclear. Due to the role of α-catenin as a mechanotransducer that recruits vinculin at cell-cell contacts, we expected α-catenin to be essential for sustaining normal levels of force transmission. Instead, using the traction force imbalance method to determine the inter-cellular force at a single cell-cell contact between cell pairs, we found that it is vinculin that is essential for sustaining normal levels of endogenous force transmission, with absence of vinculin decreasing the inter-cellular tension by over 50%. Our results constrain the potential mechanical pathways of force transmission at cell-cell contacts and suggest that vinculin can transmit forces at E-cadherin adhesions independent of α-catenin, possibly through β-catenin. Furthermore, we tested the ability of lateral cell-cell contacts to withstand external stretch and found that both vinculin and α-catenin are essential to maintain cell-cell contact stability under external forces.
    DOI:  https://doi.org/10.1016/j.bpj.2023.10.029
  10. Dev Cell. 2024 Feb 06. pii: S1534-5807(24)00037-6. [Epub ahead of print]
    Lineage motifs as developmental modules for control of cell type proportions.
    Martin Tran, Amjad Askary, Michael B Elowitz.
      In multicellular organisms, cell types must be produced and maintained in appropriate proportions. One way this is achieved is through committed progenitor cells or extrinsic interactions that produce specific patterns of descendant cell types on lineage trees. However, cell fate commitment is probabilistic in most contexts, making it difficult to infer these dynamics and understand how they establish overall cell type proportions. Here, we introduce Lineage Motif Analysis (LMA), a method that recursively identifies statistically overrepresented patterns of cell fates on lineage trees as potential signatures of committed progenitor states or extrinsic interactions. Applying LMA to published datasets reveals spatial and temporal organization of cell fate commitment in zebrafish and rat retina and early mouse embryonic development. Comparative analysis of vertebrate species suggests that lineage motifs facilitate adaptive evolutionary variation of retinal cell type proportions. LMA thus provides insight into complex developmental processes by decomposing them into simpler underlying modules.
    Keywords:  blastocyst development; cell fate correlations; cell type proportions; committed progenitor; extrinsic interactions; lineage tree; motif analysis; retina development
    DOI:  https://doi.org/10.1016/j.devcel.2024.01.017
  11. Nucleic Acids Res. 2024 Feb 14. pii: gkae085. [Epub ahead of print]
    Leveraging chromatin state transitions for the identification of regulatory networks orchestrating heart regeneration.
    Julio Cordero, Adel Elsherbiny, Yinuo Wang, Lonny Jürgensen, Florian Constanty, Stefan Günther, Melanie Boerries, Joerg Heineke, Arica Beisaw, Florian Leuschner, David Hassel, Gergana Dobreva.
      The limited regenerative capacity of the human heart contributes to high morbidity and mortality worldwide. In contrast, zebrafish exhibit robust regenerative capacity, providing a powerful model for studying how to overcome intrinsic epigenetic barriers maintaining cardiac homeostasis and initiate regeneration. Here, we present a comprehensive analysis of the histone modifications H3K4me1, H3K4me3, H3K27me3 and H3K27ac during various stages of zebrafish heart regeneration. We found a vast gain of repressive chromatin marks one day after myocardial injury, followed by the acquisition of active chromatin characteristics on day four and a transition to a repressive state on day 14, and identified distinct transcription factor ensembles associated with these events. The rapid transcriptional response involves the engagement of super-enhancers at genes implicated in extracellular matrix reorganization and TOR signaling, while H3K4me3 breadth highly correlates with transcriptional activity and dynamic changes at genes involved in proteolysis, cell cycle activity, and cell differentiation. Using loss- and gain-of-function approaches, we identified transcription factors in cardiomyocytes and endothelial cells influencing cardiomyocyte dedifferentiation or proliferation. Finally, we detected significant evolutionary conservation between regulatory regions that drive zebrafish and neonatal mouse heart regeneration, suggesting that reactivating transcriptional and epigenetic networks converging on these regulatory elements might unlock the regenerative potential of adult human hearts.
    DOI:  https://doi.org/10.1093/nar/gkae085
  12. Cell Rep. 2024 Feb 13. pii: S2211-1247(24)00110-4. [Epub ahead of print]43(2): 113782
    Unraveling the interplay between PKA inhibition and Cdk1 activation during oocyte meiotic maturation.
    Martina Santoni, Ferdinand Meneau, Nabil Sekhsoukh, Sandrine Castella, Tran Le, Marika Miot, Enrico Maria Daldello.
      Oocytes are arrested in prophase I. In vertebrates, meiotic resumption is triggered by hormonal stimulation that results in cAMP-dependent protein kinase (PKA) downregulation leading to Cdk1 activation. Yet the pathways connecting PKA to Cdk1 remain unclear. Here, we identify molecular events triggered by PKA downregulation occurring upstream of Cdk1 activation. We describe a two-step regulation controlling cyclin B1 and Mos accumulation, which depends on both translation and stabilization. Cyclin B1 accumulation is triggered by PKA inhibition upstream of Cdk1 activation, while its translation requires Cdk1 activity. Conversely, Mos translation initiates in response to the hormone, but the protein accumulates only downstream of Cdk1. Furthermore, two successive translation waves take place, the first controlled by PKA inhibition and the second by Cdk1 activation. Notably, Arpp19, an essential PKA effector, does not regulate the early PKA-dependent events. This study elucidates how PKA downregulation orchestrates multiple pathways that converge toward Cdk1 activation and induce the oocyte G2/M transition.
    Keywords:  CP: Cell biology; CP: Developmental biology; Cdk1/CyclinB; Mos-MAPK; PKA; Xenopus; meiotic maturation; oocyte; protein translation; protein turn-over
    DOI:  https://doi.org/10.1016/j.celrep.2024.113782
  13. STAR Protoc. 2024 Feb 12. pii: S2666-1667(24)00016-9. [Epub ahead of print]5(1): 102851
    Applying mechanical forces on Drosophila tissues in vivo using the StretchCo, a 3D-printable device.
    Mélanie Gracia, Bénédicte M Lefèvre, Raquel Güell Alonso, Victoire Cachoux, Maria Balakireva, Boris Guirao, Damien Cuvelier, Allison J Bardin, Yohanns Bellaïche.
      Applying mechanical forces to tissues helps to understand morphogenesis and homeostasis. Additionally, recording the dynamics of living tissues under mechanical constraints is needed to explore tissue biomechanics. Here, we present a protocol to 3D-print a StretchCo device and use it to apply uniaxial mechanical stress on the Drosophila pupal dorsal thorax epithelium. We describe steps for 3D printing, polydimethylsiloxane (PDMS) strip cutting, and glue preparation. We detail procedures for PDMS strip mounting, tissue compaction, and live imaging upon force application. For additional details on the use and execution of this protocol, please refer to Cachoux et al. (2023)1 from which the StretchCo machine has been derived.
    Keywords:  Biophysics; Developmental biology
    DOI:  https://doi.org/10.1016/j.xpro.2024.102851
  14. Curr Opin Cell Biol. 2024 Feb 09. pii: S0955-0674(24)00007-3. [Epub ahead of print]87 102328
    Nuclear mechanotransduction on skin stem cell fate regulation.
    Leah C Biggs, Yekaterina A Miroshnikova.
      Mammalian skin is a highly dynamic and regenerative organ that has long been recognized as a mechanically active composite of tissues withstanding daily compressive and tensile forces that arise from body movement. Importantly, cell- and tissue-scale mechanical signals are critical regulators of skin morphogenesis and homeostasis. These signals are sensed at the cellular periphery and transduced by mechanosensitive proteins within the plasma membrane to the cytoskeletal networks, and eventually into the nucleus to regulate chromatin organization and gene expression. The role of each of these nodes in producing a coherent mechanoresponse at both cell- and tissue-scales is emerging. Here we focus on the key cytoplasmic and nuclear mechanosensitive structures that are critical for the mammalian skin development and homeostatic maintenance. We propose that the mechanical state of the skin, in particular of its nuclear compartment, is a critical rheostat that fine-tunes developmental and homeostatic processes essential for the proper function of the organ.
    DOI:  https://doi.org/10.1016/j.ceb.2024.102328
  15. Cell Rep. 2024 Feb 12. pii: S2211-1247(24)00066-4. [Epub ahead of print]43(2): 113738
    Proline restores mitochondrial function and reverses aging hallmarks in senescent cells.
    Debanik Choudhury, Na Rong, Hamsa Vardini Senthil Kumar, Sydney Swedick, Ronel Z Samuel, Pihu Mehrotra, John Toftegaard, Nika Rajabian, Ramkumar Thiyagarajan, Ashis K Podder, Yulun Wu, Shahryar Shahini, Kenneth L Seldeen, Bruce Troen, Pedro Lei, Stelios T Andreadis.
      Mitochondrial dysfunction is a hallmark of cellular senescence, with the loss of mitochondrial function identified as a potential causal factor contributing to senescence-associated decline in cellular functions. Our recent findings revealed that ectopic expression of the pluripotency transcription factor NANOG rejuvenates dysfunctional mitochondria of senescent cells by rewiring metabolic pathways. In this study, we report that NANOG restores the expression of key enzymes, PYCR1 and PYCR2, in the proline biosynthesis pathway. Additionally, senescent mesenchymal stem cells manifest severe mitochondrial respiratory impairment, which is alleviated through proline supplementation. Proline induces mitophagy by activating AMP-activated protein kinase α and upregulating Parkin expression, enhancing mitochondrial clearance and ultimately restoring cell metabolism. Notably, proline treatment also mitigates several aging hallmarks, including DNA damage, senescence-associated β-galactosidase, inflammatory cytokine expressions, and impaired myogenic differentiation capacity. Overall, this study highlights the role of proline in mitophagy and its potential in reversing senescence-associated mitochondrial dysfunction and aging hallmarks.
    Keywords:  AMPKα; CP: Cell biology; CP: Metabolism; Parkin; aging; amino acid; autophagy; mitochondria; mitophagy; proline; senescence
    DOI:  https://doi.org/10.1016/j.celrep.2024.113738
  16. bioRxiv. 2024 Feb 01. pii: 2024.01.31.578294. [Epub ahead of print]
    NELF coordinates Pol II transcription termination and DNA replication initiation.
    Chihiro Nakayama, Yasukazu Daigaku, Yuki Aoi, Qi Fang, Hiroshi Kimura, Ali Shilatifard, Michael Tellier, Takayuki Nojima.
      Regulation of RNA polymerase II (Pol II) transcription is closely associated with cell proliferation. However, it remains unclear how the Pol II transcription program is altered in cancer to favour cell growth. Here, we find that gene expression of NELFCD , a known negative elongation factor, is up-regulated in colorectal tumours. To dissect the direct role of NELF-C on Pol II transcription in such cancer, we employed an auxin-dependent protein degradation system for NELF-C in combination with nascent transcript sequencing technologies. Strikingly, we demonstrated that the acute loss of NELF-C protein globally perturbs Pol II transcription termination and also increases transcription elongation rate, independently of promoter-proximal Pol II pausing. This results in Pol II transcription into DNA replication initiation zones, and may link to failure of the cell cycle transition into S phase. We anticipate that NELF will be a potential therapeutic target to restrict colorectal cancers by promoting transcription-replication conflict.
    HIGHLIGHTS: Expression of NELFCD transcript is up-regulated in colorectal tumors NELF-C protein is mandatory for the transition between G1-S phases during cell cycleNELF-C loss impairs transcription termination independently of Pol II promoter-proximal pausingNELF-C loss leads Pol II to invade DNA replication initiation zones.
    DOI:  https://doi.org/10.1101/2024.01.31.578294
  17. Nat Commun. 2024 Feb 15. 15(1): 1382
    Chp1 is a dedicated chaperone at the ribosome that safeguards eEF1A biogenesis.
    Melania Minoia, Jany Quintana-Cordero, Katharina Jetzinger, Ilgin Eser Kotan, Kathryn Jane Turnbull, Michela Ciccarelli, Anna E Masser, Dorina Liebers, Eloïse Gouarin, Marius Czech, Vasili Hauryliuk, Bernd Bukau, Günter Kramer, Claes Andréasson.
      Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we discover a dedicated ribosome-associated chaperone, Chp1, that rewires the cotranslational folding machinery to assist in the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). Our results indicate that during eEF1A synthesis, Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis. Aberrant eEF1A production in the absence of Chp1 triggers instant proteolysis, widespread protein aggregation, activation of Hsf1 stress transcription and compromises cellular fitness. The expression of pathogenic eEF1A2 variants linked to epileptic-dyskinetic encephalopathy is protected by Chp1. Thus, eEF1A is a difficult-to-fold protein that necessitates a biogenesis pathway starting with dedicated folding factor Chp1 at the ribosome to protect the eukaryotic cell from proteostasis collapse.
    DOI:  https://doi.org/10.1038/s41467-024-45645-w
  18. Methods Cell Biol. 2024 ;pii: S0091-679X(22)00177-7. [Epub ahead of print]182 167-185
    Cloning and expansion of repetitive DNA sequences.
    Sophie L Williams, Gideon Coster.
      Repeat and structure-prone DNA sequences comprise a large proportion of the human genome. The instability of these sequences has been implicated in a range of diseases, including cancers and neurodegenerative disorders. However, the mechanism of pathogenicity is poorly understood. As such, further studies on repetitive DNA are required. Cloning and maintaining repeat-containing substrates is challenging due to their inherent ability to form non-B DNA secondary structures which are refractory to DNA polymerases and prone to undergo rearrangements. Here, we describe an approach to clone and expand tandem-repeat DNA without interruptions, thereby allowing for its manipulation and subsequent investigation.
    Keywords:  Cloning; Genome stability; Repeats; Repetitive DNA
    DOI:  https://doi.org/10.1016/bs.mcb.2022.10.013
  19. Science. 2024 Feb 16. 383(6684): 763-770
    ATR blocks telomerase from converting DNA breaks into telomeres.
    Charles G Kinzig, George Zakusilo, Kaori K Takai, Logan R Myler, Titia de Lange.
      Telomerase, the enzyme that maintains telomeres at natural chromosome ends, should be repressed at double-strand breaks (DSBs), where neotelomere formation can cause terminal truncations. We developed an assay to detect neotelomere formation at Cas9- or I-SceI-induced DSBs in human cells. Telomerase added telomeric repeats to DSBs, leading to interstitial telomeric repeat insertions or the formation of functional neotelomeres accompanied by terminal deletions. The threat that telomerase poses to genome integrity was minimized by ataxia telangiectasia and Rad3-related (ATR) kinase signaling, which inhibited telomerase at resected DSBs. In addition to acting at resected DSBs, telomerase used the extruded strand in the Cas9 enzyme-product complex as a primer for neotelomere formation. We propose that although neotelomere formation is detrimental in normal human cells, it may allow cancer cells to escape from breakage-fusion-bridge cycles.
    DOI:  https://doi.org/10.1126/science.adg3224
  20. Acta Biomater. 2024 Feb 11. pii: S1742-7061(24)00066-7. [Epub ahead of print]
    Three dimensional fibrotic extracellular matrix directs microenvironment fiber remodeling by fibroblasts.
    Mehmet Nizamoglu, Frederique Alleblas, Taco Koster, Theo Borghuis, Judith M Vonk, Matthew J Thomas, Eric S White, Carolin K Watson, Wim Timens, Karim C El Kasmi, Barbro N Melgert, Irene H Heijink, Janette K Burgess.
      Idiopathic pulmonary fibrosis (IPF), for which effective treatments are limited, results in excessive and disorganized deposition of aberrant extracellular matrix (ECM). An altered ECM microenvironment is postulated to contribute to disease progression through inducing profibrotic behavior of lung fibroblasts, the main producers and regulators of ECM. Here, we examined this hypothesis in a 3D in vitro model system by growing primary human lung fibroblasts in ECM-derived hydrogels from non-fibrotic (control) or IPF lung tissue. Using this model, we compared how control and IPF lung-derived fibroblasts responded in control and fibrotic microenvironments in a combinatorial manner. Culture of fibroblasts in fibrotic hydrogels did not alter in the overall amount of collagen or glycosaminoglycans but did cause a drastic change in fiber organization compared to culture in control hydrogels. High-density collagen percentage was increased by control fibroblasts in IPF hydrogels at day 7, but decreased at day 14. In contrast, IPF fibroblasts only decreased the high-density collagen percentage at day 14, which was accompanied by enhanced fiber alignment in IPF hydrogels. Similarly, stiffness of fibrotic hydrogels was increased only by control fibroblasts by day 14 while those of control hydrogels were not altered by fibroblasts. These data highlight how the ECM-remodeling responses of fibroblasts are influenced by the origin of both the cells and the ECM. Moreover, by showing how the 3D microenvironment plays a crucial role in directing cells, our study paves the way in guiding future investigations examining fibrotic processes with respect to ECM remodeling responses of fibroblasts. STATEMENT OF SIGNIFICANCE: In this study, we investigated the influence of the altered extracellular matrix (ECM) in Idiopathic Pulmonary Fibrosis (IPF), using a 3D in vitro model system composed of ECM-derived hydrogels from both IPF and control lungs, seeded with human IPF and control lung fibroblasts. While our results indicated that fibrotic microenvironment did not change the overall collagen or glycosaminoglycan content, it resulted in a dramatically alteration of fiber organization and mechanical properties. Control fibroblasts responded differently from IPF fibroblasts, highlighting the unique instructive role of the fibrotic ECM and the interplay with fibroblast origin. These results underscore the importance of 3D microenvironments in guiding pro-fibrotic responses, offering potential insights for future IPF therapies as well as other fibrotic diseases and cancer.
    Keywords:  ECM-derived hydrogels; biomechanics; collagen organization; crosslinking; fibrosis; idiopathic pulmonary fibrosis
    DOI:  https://doi.org/10.1016/j.actbio.2024.02.008
  21. Dev Cell. 2024 Feb 02. pii: S1534-5807(24)00031-5. [Epub ahead of print]
    Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition.
    John-Poul Ng-Blichfeldt, Benjamin J Stewart, Menna R Clatworthy, Julie M Williams, Katja Röper.
      During kidney development, nephron epithelia arise de novo from fate-committed mesenchymal progenitors through a mesenchymal-to-epithelial transition (MET). Downstream of fate specification, transcriptional mechanisms that drive establishment of epithelial morphology are poorly understood. We used human iPSC-derived renal organoids, which recapitulate nephrogenesis, to investigate mechanisms controlling renal MET. Multi-ome profiling via snRNA-seq and ATAC-seq of organoids identified dynamic changes in gene expression and chromatin accessibility driven by activators and repressors throughout MET. CRISPR interference identified that paired box 8 (PAX8) is essential for initiation of MET in human renal organoids, contrary to in vivo mouse studies, likely by activating a cell-adhesion program. While Wnt/β-catenin signaling specifies nephron fate, we find that it must be attenuated to allow hepatocyte nuclear factor 1-beta (HNF1B) and TEA-domain(TEAD) transcription factors to drive completion of MET. These results identify the interplay between fate commitment and morphogenesis in the developing human kidney, with implications for understanding both developmental kidney diseases and aberrant epithelial plasticity following adult renal tubular injury.
    Keywords:  epithelial polarity; kidney development; mesenchymal-to-epithelial transition; organoids; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.devcel.2024.01.011
  22. Nat Commun. 2024 Feb 12. 15(1): 1311
    In situ structure of actin remodeling during glucose-stimulated insulin secretion using cryo-electron tomography.
    Weimin Li, Angdi Li, Bing Yu, Xiaoxiao Zhang, Xiaoyan Liu, Kate L White, Raymond C Stevens, Wolfgang Baumeister, Andrej Sali, Marion Jasnin, Liping Sun.
      Actin mediates insulin secretion in pancreatic β-cells through remodeling. Hampered by limited resolution, previous studies have offered an ambiguous depiction as depolymerization and repolymerization. We report the in situ structure of actin remodeling in INS-1E β-cells during glucose-stimulated insulin secretion at nanoscale resolution. After remodeling, the actin filament network at the cell periphery exhibits three marked differences: 12% of actin filaments reorient quasi-orthogonally to the ventral membrane; the filament network mainly remains as cell-stabilizing bundles but partially reconfigures into a less compact arrangement; actin filaments anchored to the ventral membrane reorganize from a "netlike" to a "blooming" architecture. Furthermore, the density of actin filaments and microtubules around insulin secretory granules decreases, while actin filaments and microtubules become more densely packed. The actin filament network after remodeling potentially precedes the transport and release of insulin secretory granules. These findings advance our understanding of actin remodeling and its role in glucose-stimulated insulin secretion.
    DOI:  https://doi.org/10.1038/s41467-024-45648-7
  23. Methods Cell Biol. 2024 ;pii: S0091-679X(22)00176-5. [Epub ahead of print]182 21-33
    Generation of aneuploid cells and assessment of their ability to survive in presence of chemotherapeutic agents.
    Marica Rosaria Ippolito, Stefano Santaguida.
      Aneuploidy is a condition in which cells have an abnormal number of chromosomes that is not a multiple of the haploid complement. It is known that aneuploidy has detrimental consequences on cell physiology, such as genome instability, metabolic and proteotoxic stress and decreased cellular fitness. Importantly, aneuploidy is a hallmark of tumors and it is associated with resistance to chemotherapeutic agents and poor clinical outcome. To shed light into how aneuploidy contributes to chemoresistance, we induced chromosome mis-segregation in human cancer cell lines, then treated them with several chemotherapeutic agents and evaluated the emergence of chemoresistance. By doing so, we found that elevation of chromosome mis-segregation promotes resistance to chemotherapeutic agents through the expansion of aneuploid karyotypes and subsequent selection of specific aneuploidies essential for cellular viability under those stressful conditions. Here, we describe a method to generate aneuploid cell populations and to evaluate their resistance to anti-cancer agents. This protocol has been already successfully employed and can be further utilized to accelerate the exploration of the role of aneuploidy in chemoresistance.
    Keywords:  Aneuploidy; Chemoresistance; Genome instability; Mitotic errors; Mps1
    DOI:  https://doi.org/10.1016/bs.mcb.2022.10.012
  24. Nature. 2024 Feb 14.
    Genetic determinants of micronucleus formation in vivo.
    Sanger Mouse Genetics Project
      Genomic instability arising from defective responses to DNA damage1 or mitotic chromosomal imbalances2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR-Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology1.
    DOI:  https://doi.org/10.1038/s41586-023-07009-0
  25. Nat Genet. 2024 Feb 15.
    Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility.
    Satoko Ishii, Taishi Kakizuka, Sung-Joon Park, Ayako Tagawa, Chiaki Sanbo, Hideyuki Tanabe, Yasuyuki Ohkawa, Mahito Nakanishi, Kenta Nakai, Yusuke Miyanari.
      Chromatin accessibility is a hallmark of active regulatory regions and is functionally linked to transcriptional networks and cell identity. However, the molecular mechanisms and networks that govern chromatin accessibility have not been thoroughly studied. Here we conducted a genome-wide CRISPR screening combined with an optimized ATAC-see protocol to identify genes that modulate global chromatin accessibility. In addition to known chromatin regulators like CREBBP and EP400, we discovered a number of previously unrecognized proteins that modulate chromatin accessibility, including TFDP1, HNRNPU, EIF3D and THAP11 belonging to diverse biological pathways. ATAC-seq analysis upon their knockouts revealed their distinct and specific effects on chromatin accessibility. Remarkably, we found that TFDP1, a transcription factor, modulates global chromatin accessibility through transcriptional regulation of canonical histones. In addition, our findings highlight the manipulation of chromatin accessibility as an approach to enhance various cell engineering applications, including genome editing and induced pluripotent stem cell reprogramming.
    DOI:  https://doi.org/10.1038/s41588-024-01658-1
  26. Sci Adv. 2024 Feb 16. 10(7): eadk0639
    Biphasic regulation of epigenetic state by matrix stiffness during cell reprogramming.
    Yang Song, Jennifer Soto, Sze Yue Wong, Yifan Wu, Tyler Hoffman, Navied Akhtar, Sam Norris, Julia Chu, Hyungju Park, Douglas O Kelkhoff, Cheen Euong Ang, Marius Wernig, Andrea Kasko, Timothy L Downing, Mu-Ming Poo, Song Li.
      We investigate how matrix stiffness regulates chromatin reorganization and cell reprogramming and find that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, we observe peak levels of histone acetylation and histone acetyltransferase (HAT) activity in the nucleus on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, the cotransporters shuttling HAT into the nucleus, rises with decreasing matrix stiffness; however, reduced importin-9 on soft matrices limits nuclear transport. These two factors result in a biphasic regulation of HAT transport into nucleus, which is directly demonstrated on matrices with dynamically tunable stiffness. Our findings unravel a mechanism of the mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.
    DOI:  https://doi.org/10.1126/sciadv.adk0639
  27. Nature. 2024 Feb 14.
    A single-cell time-lapse of mouse prenatal development from gastrula to birth.
    Chengxiang Qiu, Beth K Martin, Ian C Welsh, Riza M Daza, Truc-Mai Le, Xingfan Huang, Eva K Nichols, Megan L Taylor, Olivia Fulton, Diana R O'Day, Anne Roshella Gomes, Saskia Ilcisin, Sanjay Srivatsan, Xinxian Deng, Christine M Disteche, William Stafford Noble, Nobuhiko Hamazaki, Cecilia B Moens, David Kimelman, Junyue Cao, Alexander F Schier, Malte Spielmann, Stephen A Murray, Cole Trapnell, Jay Shendure.
      The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.
    DOI:  https://doi.org/10.1038/s41586-024-07069-w
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