bims-ginsta Biomed News
on Genome instability
Issue of 2024–03–17
twenty-six papers selected by
Jinrong Hu, National University of Singapore
  1. Control of poly(A)-tail length and translation in vertebrate oocytes and early embryos.
  2. Sister chromatid cohesion establishment during DNA replication termination.
  3. Spatially organized cellular communities form the developing human heart.
  4. Mechanosensing regulates tissue repair program in macrophages.
  5. Two distinct waves of transcriptome and translatome changes drive Drosophila germline stem cell differentiation.
  6. Reduction of cortical pulling at mitotic entry facilitates aster centration.
  7. Female fertility and the mammalian egg's zona pellucida.
  8. YTHDF2 governs muscle size through a targeted modulation of proteostasis.
  9. Dynamic microenvironments shape nuclear organization and gene expression.
  10. Doublecortin reinforces microtubules to promote growth cone advance in soft environments.
  11. Insulin signaling regulates R2 retrotransposon expression to orchestrate transgenerational rDNA copy number maintenance.
  12. A BMP-controlled metabolic-epigenetic signaling cascade directs midfacial morphogenesis.
  13. Mitochondrial complex I activity in microglia sustains neuroinflammation.
  14. The PCM scaffold enables RNA localization to centrosomes.
  15. Broken chromosomes heading into mitosis: More than one way to patch a flat tire.
  16. ATR limits Rad18-mediated PCNA monoubiquitination to preserve replication fork and telomerase-independent telomere stability.
  17. Efficient and reproducible generation of human iPSC-derived cardiomyocytes using a stirred bioreactor.
  18. Tyrosine 1-phosphorylated RNA polymerase II transcribes PROMPTs to facilitate proximal promoter pausing and induce global transcriptional repression in response to DNA damage.
  19. Adult and neonatal models of chemogenetic heart failure caused by oxidative stress.
  20. Age-dependent loss of HAPLN1 erodes vascular integrity via indirect upregulation of endothelial ICAM1 in melanoma.
  21. Short cell cycle duration is a phenotype of human epidermal stem cells.
  22. ZNF827 is a single-stranded DNA binding protein that regulates the ATR-CHK1 DNA damage response pathway.
  23. Liprin-α proteins are master regulators of human presynapse assembly.
  24. MYC activates transcriptional enhancers to drive cancer progression.
  25. N-cadherin dynamically regulates pediatric glioma cell migration in complex environments.
  26. Cytoplasmic retention of the DNA/RNA-binding protein FUS ameliorates organ fibrosis in mice.
  1. Dev Cell. 2024 Mar 05. pii: S1534-5807(24)00105-9. [Epub ahead of print]
    Control of poly(A)-tail length and translation in vertebrate oocytes and early embryos.
    Kehui Xiang, Jimmy Ly, David P Bartel.
      During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated has been unclear. Here, we performed tail-length and translational profiling of mRNA reporter libraries (each with millions of 3' UTR sequence variants) in frog oocytes and embryos and in fish embryos. Contrasting to previously proposed cytoplasmic polyadenylation elements (CPEs), we found that a shorter element, UUUUA, together with the polyadenylation signal (PAS), specify cytoplasmic polyadenylation, and we identified contextual features that modulate the activity of both elements. In maturing oocytes, this tail lengthening occurs against a backdrop of global deadenylation and the action of C-rich elements that specify tail-length-independent translational repression. In embryos, cytoplasmic polyadenylation becomes more permissive, and additional elements specify waves of stage-specific deadenylation. Together, these findings largely explain the complex tapestry of tail-length changes observed in early frog and fish development, with strong evidence of conservation in both mice and humans.
    Keywords:  3' UTR; CPE; CPEB; cytoplasmic polyadenylation; deadenylation; embryonic development; gene regulation; oocyte maturation; poly(A) tails; translation regulation
    DOI:  https://doi.org/10.1016/j.devcel.2024.02.007
  2. Science. 2024 Mar 14. eadf0224
    Sister chromatid cohesion establishment during DNA replication termination.
    George Cameron, Dominika T Gruszka, Rhian Gruar, Sherry Xie, Çağla Kaya, Kim A Nasmyth, Jonathan Baxter, Madhusudhan Srinivasan, Hasan Yardimci.
      Newly copied sister chromatids are tethered together by the cohesin complex, but how sister chromatid cohesion is coordinated with DNA replication is poorly understood. Prevailing models suggest cohesin complexes, bound to DNA before replication, remain behind the advancing replication fork to keep sister chromatids together. By visualizing single replication forks colliding with pre-loaded cohesin complexes, we find that the replisome instead pushes cohesin to where a converging replisome is met. While the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA and provides cohesion. Additionally, we show that CMG disassembly during replication termination is vital for proper cohesion in budding yeast. Together, our results support a new model where sister chromatid cohesion is established during DNA replication termination.
    DOI:  https://doi.org/10.1126/science.adf0224
  3. Nature. 2024 Mar 13.
    Spatially organized cellular communities form the developing human heart.
    Elie N Farah, Robert K Hu, Colin Kern, Qingquan Zhang, Ting-Yu Lu, Qixuan Ma, Shaina Tran, Bo Zhang, Daniel Carlin, Alexander Monell, Andrew P Blair, Zilu Wang, Jacqueline Eschbach, Bin Li, Eugin Destici, Bing Ren, Sylvia M Evans, Shaochen Chen, Quan Zhu, Neil C Chi.
      The heart, which is the first organ to develop, is highly dependent on its form to function1,2. However, how diverse cardiac cell types spatially coordinate to create the complex morphological structures that are crucial for heart function remains unclear. Here we integrated single-cell RNA-sequencing with high-resolution multiplexed error-robust fluorescence in situ hybridization to resolve the identity of the cardiac cell types that develop the human heart. This approach also provided a spatial mapping of individual cells that enables illumination of their organization into cellular communities that form distinct cardiac structures. We discovered that many of these cardiac cell types further specified into subpopulations exclusive to specific communities, which support their specialization according to the cellular ecosystem and anatomical region. In particular, ventricular cardiomyocyte subpopulations displayed an unexpected complex laminar organization across the ventricular wall and formed, with other cell subpopulations, several cellular communities. Interrogating cell-cell interactions within these communities using in vivo conditional genetic mouse models and in vitro human pluripotent stem cell systems revealed multicellular signalling pathways that orchestrate the spatial organization of cardiac cell subpopulations during ventricular wall morphogenesis. These detailed findings into the cellular social interactions and specialization of cardiac cell types constructing and remodelling the human heart offer new insights into structural heart diseases and the engineering of complex multicellular tissues for human heart repair.
    DOI:  https://doi.org/10.1038/s41586-024-07171-z
  4. Sci Adv. 2024 Mar 15. 10(11): eadk6906
    Mechanosensing regulates tissue repair program in macrophages.
    Matthew L Meizlish, Yoshitaka Kimura, Scott D Pope, Rita Matta, Catherine Kim, Naomi H Philip, Linde Meyaard, Anjelica Gonzalez, Ruslan Medzhitov.
      Tissue-resident macrophages play important roles in tissue homeostasis and repair. However, how macrophages monitor and maintain tissue integrity is not well understood. The extracellular matrix (ECM) is a key structural and organizational component of all tissues. Here, we find that macrophages sense the mechanical properties of the ECM to regulate a specific tissue repair program. We show that macrophage mechanosensing is mediated by cytoskeletal remodeling and can be performed in three-dimensional environments through a noncanonical, integrin-independent mechanism analogous to amoeboid migration. We find that these cytoskeletal dynamics also integrate biochemical signaling by colony-stimulating factor 1 and ultimately regulate chromatin accessibility to control the mechanosensitive gene expression program. This study identifies an "amoeboid" mode of ECM mechanosensing through which macrophages may regulate tissue repair and fibrosis.
    DOI:  https://doi.org/10.1126/sciadv.adk6906
  5. EMBO J. 2024 Mar 13.
    Two distinct waves of transcriptome and translatome changes drive Drosophila germline stem cell differentiation.
    Tamsin J Samuels, Jinghua Gui, Daniel Gebert, Felipe Karam Teixeira.
      The tight control of fate transitions during stem cell differentiation is essential for proper tissue development and maintenance. However, the challenges in studying sparsely distributed adult stem cells in a systematic manner have hindered efforts to identify how the multilayered regulation of gene expression programs orchestrates stem cell differentiation in vivo. Here, we synchronised Drosophila female germline stem cell (GSC) differentiation in vivo to perform in-depth transcriptome and translatome analyses at high temporal resolution. This characterisation revealed widespread and dynamic changes in mRNA level, promoter usage, exon inclusion, and translation efficiency. Transient expression of the master regulator, Bam, drives a first wave of expression changes, primarily modifying the cell cycle program. Surprisingly, as Bam levels recede, differentiating cells return to a remarkably stem cell-like transcription and translation program, with a few crucial changes feeding into a second phase driving terminal differentiation to form the oocyte. Altogether, these findings reveal that rather than a unidirectional accumulation of changes, the in vivo differentiation of stem cells relies on distinctly regulated and developmentally sequential waves.
    Keywords:  Differentiation; Drosophila; Germline; Transcription; Translation
    DOI:  https://doi.org/10.1038/s44318-024-00070-z
  6. J Cell Sci. 2024 Mar 12. pii: jcs.262037. [Epub ahead of print]
    Reduction of cortical pulling at mitotic entry facilitates aster centration.
    Anne Rosfelter, Ghislain de Labbey, Janet Chenevert, Rémi Dumollard, Sebastien Schaub, Zoltan Machaty, Lydia Besnardeau, Daniel Gonzalez Suarez, Céline Hebras, Hervé Turlier, David Burgess, Alex McDougall.
      Equal cell division relies upon astral microtubule-based centering mechanisms, yet how the interplay between mitotic entry, cortical force generation, and long astral microtubules leads to symmetric cell division is not resolved. We report that a cortically-located sperm aster displaying long astral microtubules that penetrate the whole zygote does not undergo centration until mitotic entry. At mitotic entry we find that microtubule-based cortical pulling is lost. Quantitative measurements of cortical pulling and cytoplasmic pulling together with physical simulations suggested a wavelike loss of cortical pulling at mitotic entry led to aster centration based on cytoplasmic pulling. Cortical actin is lost from the cortex at mitotic entry coincident with a fall in cortical tension from around 300pN/µm to 100pN/µm. Following the loss of cortical force generators at mitotic entry long microtubule-based cytoplasmic pulling is sufficient to displace the aster towards the cell center. These data reveal how mitotic aster centration is coordinated with mitotic entry in chordate zygotes.
    Keywords:  Cell cycle; Mitotic apparatus; Nuclear migration; Sperm aster
    DOI:  https://doi.org/10.1242/jcs.262037
  7. Histol Histopathol. 2024 Mar 04. 18728
    Female fertility and the mammalian egg's zona pellucida.
    Paul M Wassarman, Eveline S Litscher.
      All mammalian eggs are surrounded by a relatively thick extracellular matrix (ECM) or zona pellucida (ZP) to which free-swimming sperm bind in a species-restricted manner during fertilization. The ZP consists of either three (e.g., Mus musculus) or four (e.g., Homo sapiens) glycosylated proteins, called ZP1-4. These proteins are unlike those found in somatic cell ECM, are encoded by single-copy genes on different chromosomes, and are well conserved among different mammals. Mammalian ZP proteins are synthesized as polypeptide precursors by growing oocytes that will become ovulated, unfertilized eggs. These precursors are processed to remove a signal-sequence and carboxy-terminal propeptide and are secreted into the extracellular space. Secreted ZP proteins assemble into long, crosslinked filaments that exhibit a structural repeat due to the presence of ZP2-ZP3 dimers every 140 Å or so along filaments. Filaments are crosslinked by ZP1 and are oriented either perpendicular, parallel, or randomly to the plasma membrane of eggs depending on their position in the ZP. Free-swimming mouse sperm recognize and bind to ZP2 or ZP3 that serve as sperm receptors. Acrosome-intact sperm bind to ZP3 oligosaccharides and acrosome-reacted sperm bind to ZP2 polypeptide. ZP filaments fail to assemble in the absence of either nascent ZP2 or ZP3 and results in mouse eggs that lack a ZP and female infertility. Gene sequence variations due to point, missense, or frameshift mutations in genes encoding ZP1-4 results in human eggs that lack a ZP or have an abnormal ZP and female infertility. These and other features of the mouse and human egg's ZP are discussed here.
    DOI:  https://doi.org/10.14670/HH-18-728
  8. Nat Commun. 2024 Mar 11. 15(1): 2176
    YTHDF2 governs muscle size through a targeted modulation of proteostasis.
    Christopher J Gilbert, Charles P Rabolli, Volha A Golubeva, Kristina M Sattler, Meifang Wang, Arsh Ketabforoush, W David Arnold, Christoph Lepper, Federica Accornero.
      The regulation of proteostasis is fundamental for maintenance of muscle mass and function. Activation of the TGF-β pathway drives wasting and premature aging by favoring the proteasomal degradation of structural muscle proteins. Yet, how this critical post-translational mechanism is kept in check to preserve muscle health remains unclear. Here, we reveal the molecular link between the post-transcriptional regulation of m6A-modified mRNA and the modulation of SMAD-dependent TGF-β signaling. We show that the m6A-binding protein YTHDF2 is essential to determining postnatal muscle size. Indeed, muscle-specific genetic deletion of YTHDF2 impairs skeletal muscle growth and abrogates the response to hypertrophic stimuli. We report that YTHDF2 controls the mRNA stability of the ubiquitin ligase ASB2 with consequences on anti-growth gene program activation through SMAD3. Our study identifies a post-transcriptional to post-translational mechanism for the coordination of gene expression in muscle.
    DOI:  https://doi.org/10.1038/s41467-024-46546-8
  9. Curr Opin Genet Dev. 2024 Mar 09. pii: S0959-437X(24)00026-1. [Epub ahead of print]86 102177
    Dynamic microenvironments shape nuclear organization and gene expression.
    Gabriela Hayward-Lara, Matthew D Fischer, Mustafa Mir.
      Live imaging has revealed that the regulation of gene expression is largely driven by transient interactions. For example, many regulatory proteins bind chromatin for just seconds, and loop-like genomic contacts are rare and last only minutes. These discoveries have been difficult to reconcile with our canonical models that are predicated on stable and hierarchical interactions. Proteomic microenvironments that concentrate nuclear factors may explain how brief interactions can still mediate gene regulation by creating conditions where reactions occur more frequently. Here, we summarize new imaging technologies and recent discoveries implicating microenvironments as a potential driver of nuclear function. Finally, we propose that key properties of proteomic microenvironments, such as their size, enrichment, and lifetimes, are directly linked to regulatory function.
    DOI:  https://doi.org/10.1016/j.gde.2024.102177
  10. bioRxiv. 2024 Feb 29. pii: 2024.02.28.582626. [Epub ahead of print]
    Doublecortin reinforces microtubules to promote growth cone advance in soft environments.
    Alessandro Dema, Rabab A Charafeddine, Jeffrey van Haren, Shima Rahgozar, Giulia Viola, Kyle A Jacobs, Matthew L Kutys, Torsten Wittmann.
      Doublecortin (DCX) is a microtubule-associated protein critical for brain development. Although most highly expressed in the developing central nervous system, the molecular function of DCX in neuron morphogenesis remains unknown and controversial. We demonstrate that DCX function is intimately linked to its microtubule-binding activity. By using human induced pluripotent stem cell (hiPSC)- derived cortical i 3 Neurons genome engineered to express mEmerald-tagged DCX from the endogenous locus, we find that DCX-MT interactions become highly polarized very early during neuron morphogenesis. DCX becomes enriched only on straight microtubules in advancing growth cones with approximately 120 DCX molecules bound per micrometer of growth cone microtubule. At a similar saturation, microtubule-bound DCX molecules begin to impede lysosome transport, and thus can potentially control growth cone organelle entry. In addition, by comparing control, DCX-mEmerald and knockout DCX -/Y i 3 Neurons, we find that DCX stabilizes microtubules in the growth cone peripheral domain by reducing the microtubule catastrophe frequency and the depolymerization rate. DCX -/Y i 3 Neuron morphogenesis was inhibited in soft microenvironments that mimic the viscoelasticity of brain tissue and DCX -/Y neurites failed to grow toward brain-derived neurotrophic factor (BDNF) gradients. Together with high resolution traction force microscopy data, we propose a model in which DCX-decorated, rigid growth cone microtubules provide intracellular mechanical resistance to actomyosin generated contractile forces in soft physiological environments in which weak and transient adhesion-mediated forces in the growth cone periphery may be insufficient for productive growth cone advance. These data provide a new mechanistic understanding of how DCX mutations cause lissencephaly-spectrum brain malformations by impacting growth cone dynamics during neuron morphogenesis in physiological environments.
    DOI:  https://doi.org/10.1101/2024.02.28.582626
  11. bioRxiv. 2024 Feb 29. pii: 2024.02.28.582629. [Epub ahead of print]
    Insulin signaling regulates R2 retrotransposon expression to orchestrate transgenerational rDNA copy number maintenance.
    Jonathan O Nelson, Alyssa Slicko, Amelie A Raz, Yukiko M Yamashita.
      Preserving a large number of essential yet highly unstable ribosomal DNA (rDNA) repeats is critical for the germline to perpetuate the genome through generations. Spontaneous rDNA loss must be countered by rDNA copy number (CN) expansion. Germline rDNA CN expansion is best understood in Drosophila melanogaster , which relies on unequal sister chromatid exchange (USCE) initiated by DNA breaks at rDNA. The rDNA-specific retrotransposon R2 responsible for USCE-inducing DNA breaks is typically expressed only when rDNA CN is low to minimize the danger of DNA breaks; however, the underlying mechanism of R2 regulation remains unclear. Here we identify the insulin receptor (InR) as a major repressor of R2 expression, limiting unnecessary R2 activity. Through single-cell RNA sequencing we find that male germline stem cells (GSCs), the major cell type that undergoes rDNA CN expansion, have reduced InR expression when rDNA CN is low. Reduced InR activity in turn leads to R2 expression and CN expansion. We further find that dietary manipulation alters R2 expression and rDNA CN expansion activity. This work reveals that the insulin pathway integrates rDNA CN surveying with environmental sensing, revealing a potential mechanism by which diet exerts heritable changes to genomic content.
    DOI:  https://doi.org/10.1101/2024.02.28.582629
  12. J Clin Invest. 2024 Mar 11. pii: e165787. [Epub ahead of print]
    A BMP-controlled metabolic-epigenetic signaling cascade directs midfacial morphogenesis.
    Jingwen Yang, Lingxin Zhu, Haichun Pan, Hiroki Ueharu, Masako Toda, Qian Yang, Shawn A Hallett, Lorin E Olson, Yuji Mishina.
      Craniofacial anomalies, especially midline facial defects, are among the most common birth defects in patients associated with increased mortality or require lifelong treatment. During mammalian embryogenesis, specific instructions arising at genetic, signaling, and metabolic levels are important for stem cell behaviors and fate determination, but how these functionally relevant mechanisms are coordinated to regulate craniofacial morphogenesis remain unknown. Here, we report that BMP signaling in cranial neural crest cells (CNCCs) is critical for glycolytic lactate production and subsequent epigenetic histone lactylation, thereby dictating craniofacial morphogenesis. Elevated BMP signaling in CNCCs through constitutively activated ACVR1 (ca-ACVR1) suppressed glycolytic activity and blocked lactate production via a p53-dependent process that resulted in severe midline facial defects. By modulating epigenetic remodeling, BMP signaling-dependent lactate generation drived histone lactylation levels to alter essential genes of Pdgfra thus regulating CNCC behavior in vitro as well as in vivo. These findings define an axis wherein the BMP signaling controls a metabolic-epigenetic cascade to direct craniofacial morphogenesis, thus providing a conceptual framework for understanding the interaction between genetic and metabolic cues operative during embryonic development. These findings indicate potential preventive strategies of congenital craniofacial birth defects via modulating metabolic-driven histone lactylation.
    Keywords:  Development; Embryonic development; Mouse models; Therapeutics
    DOI:  https://doi.org/10.1172/JCI165787
  13. Nature. 2024 Mar 13.
    Mitochondrial complex I activity in microglia sustains neuroinflammation.
    L Peruzzotti-Jametti, C M Willis, G Krzak, R Hamel, L Pirvan, R-B Ionescu, J A Reisz, H A Prag, M E Garcia-Segura, V Wu, Y Xiang, B Barlas, A M Casey, A M R van den Bosch, A M Nicaise, L Roth, G R Bates, H Huang, P Prasad, A E Vincent, C Frezza, C Viscomi, G Balmus, Z Takats, J C Marioni, A D'Alessandro, M P Murphy, I Mohorianu, S Pluchino.
      Sustained smouldering, or low-grade activation, of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However, how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here, using a multiomics approach, we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically, blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system3.
    DOI:  https://doi.org/10.1038/s41586-024-07167-9
  14. bioRxiv. 2024 Jan 14. pii: 2024.01.13.575509. [Epub ahead of print]
    The PCM scaffold enables RNA localization to centrosomes.
    Junnan Fang, Weiyi Tian, Melissa A Quintanilla, Jordan R Beach, Dorothy A Lerit.
      As microtubule-organizing centers, centrosomes direct assembly of the bipolar mitotic spindle required for chromosome segregation and genome stability. Centrosome activity requires the dynamic assembly of pericentriolar material (PCM), the composition and organization of which changes throughout the cell cycle. Recent studies highlight the conserved localization of several mRNAs encoded from centrosome-associated genes enriched at centrosomes, including Pericentrin-like protein ( Plp ) mRNA. However, relatively little is known about how RNAs localize to centrosomes and influence centrosome function. Here, we examine mechanisms underlying the subcellular localization of Plp mRNA. We find that Plp mRNA localization is puromycin-sensitive, and the Plp coding sequence is both necessary and sufficient for RNA localization, consistent with a co-translational transport mechanism. We identify regions within the Plp coding sequence that regulate Plp mRNA localization. Finally, we show that protein-protein interactions critical for elaboration of the PCM scaffold permit RNA localization to centrosomes. Taken together, these findings inform the mechanistic basis of Plp mRNA localization and lend insight into the oscillatory enrichment of RNA at centrosomes.
    DOI:  https://doi.org/10.1101/2024.01.13.575509
  15. J Cell Biol. 2024 Apr 01. pii: e202401085. [Epub ahead of print]223(4):
    Broken chromosomes heading into mitosis: More than one way to patch a flat tire.
    C Luke Messer, Donald T Fox.
      A cell dealing with a broken chromosome in mitosis is like a driver dealing with a flat tire on the highway: damage repair must occur under non-ideal circumstances. Mitotic chromosome breaks encounter problems related to structures called micronuclei. These aberrant nuclei are linked to cell death, mutagenesis, and cancer. In the last few years, a flurry of studies illuminated two mechanisms that prevent mitotic problems related to micronuclei. One mechanism prevents micronuclei from forming during mitosis and involves DNA Polymerase Theta, a DNA repair regulator that patches up broken mitotic chromosomes. A second mechanism is activated after micronuclei form and then rupture, and involves CIP2A and TOPBP1 proteins, which patch micronuclear fragments to promote their subsequent mitotic segregation. Here, we review recent progress in this field of mitotic DNA damage and discuss why multiple mechanisms exist. Future studies in this exciting area will reveal new DNA break responses and inform therapeutic strategies.
    DOI:  https://doi.org/10.1083/jcb.202401085
  16. EMBO J. 2024 Mar 11.
    ATR limits Rad18-mediated PCNA monoubiquitination to preserve replication fork and telomerase-independent telomere stability.
    Siyuan Chen, Chen Pan, Jun Huang, Ting Liu.
      Upon replication fork stalling, the RPA-coated single-stranded DNA (ssDNA) formed behind the fork activates the ataxia telangiectasia-mutated and Rad3-related (ATR) kinase, concomitantly initiating Rad18-dependent monoubiquitination of PCNA. However, whether crosstalk exists between these two events and the underlying physiological implications of this interplay remain elusive. In this study, we demonstrate that during replication stress, ATR phosphorylates human Rad18 at Ser403, an adjacent residue to a previously unidentified PIP motif (PCNA-interacting peptide) within Rad18. This phosphorylation event disrupts the interaction between Rad18 and PCNA, thereby restricting the extent of Rad18-mediated PCNA monoubiquitination. Consequently, excessive accumulation of the tumor suppressor protein SLX4, now characterized as a novel reader of ubiquitinated PCNA, at stalled forks is prevented, contributing to the prevention of stalled fork collapse. We further establish that ATR preserves telomere stability in alternative lengthening of telomere (ALT) cells by restricting Rad18-mediated PCNA monoubiquitination and excessive SLX4 accumulation at telomeres. These findings shed light on the complex interplay between ATR activation, Rad18-dependent PCNA monoubiquitination, and SLX4-associated stalled fork processing, emphasizing the critical role of ATR in preserving replication fork stability and facilitating telomerase-independent telomere maintenance.
    Keywords:  ATR; Fork Collapse; PCNA Monoubiquitination; RAD18; Telomere Stability
    DOI:  https://doi.org/10.1038/s44318-024-00066-9
  17. bioRxiv. 2024 Feb 28. pii: 2024.02.24.581789. [Epub ahead of print]
    Efficient and reproducible generation of human iPSC-derived cardiomyocytes using a stirred bioreactor.
    Maksymilian Prondzynski, Raul H Bortolin, Paul Berkson, Michael A Trembley, Kevin Shani, Mason E Sweat, Joshua Mayourian, Albert M Cordoves, Nnaemeka J Anyanwu, Yashasvi Tharani, Justin Cotton, Joseph B Milosh, David Walker, Yan Zhang, Fujian Liu, Xujie Liu, Kevin K Parker, Vassilios J Bezzerides, William T Pu.
      In the last decade human iPSC-derived cardiomyocytes (hiPSC-CMs) proved to be valuable for cardiac disease modeling and cardiac regeneration, yet challenges with scale, quality, inter-batch consistency, and cryopreservation remain, reducing experimental reproducibility and limiting clinical translation. Here, we report a robust cardiac differentiation protocol that uses Wnt modulation and a stirred suspension bioreactor to produce on average 124 million hiPSC-CMs with >90% purity using a variety of hiPSC lines (19 differentiations; 10 iPSC lines). After controlled freeze and thaw, bioreactor-derived CMs (bCMs) showed high viability (>90%), interbatch reproducibility in cellular morphology, function, drug response and ventricular identity, which was further supported by single cell transcriptomes. bCMs on microcontact printed substrates revealed a higher degree of sarcomere maturation and viability during long-term culture compared to monolayer-derived CMs (mCMs). Moreover, functional investigation of bCMs in 3D engineered heart tissues showed earlier and stronger force production during long-term culture, and robust pacing capture up to 4 Hz when compared to mCMs. bCMs derived from this differentiation protocol will expand the applications of hiPSC-CMs by providing a reproducible, scalable, and resource efficient method to generate cardiac cells with well-characterized structural and functional properties superior to standard mCMs.
    DOI:  https://doi.org/10.1101/2024.02.24.581789
  18. Genome Res. 2024 Mar 11.
    Tyrosine 1-phosphorylated RNA polymerase II transcribes PROMPTs to facilitate proximal promoter pausing and induce global transcriptional repression in response to DNA damage.
    Kamal Ajit, Adele Alagia, Kaspar Burger, Monika Gullerova.
      DNA damage triggers a complex transcriptional response that involves both activation and repression of gene expression. In this study, we investigated global changes in transcription in response to ionizing irradiation (IR), which induces double-strand breaks in DNA. We used mNET-seq to profile nascent transcripts bound to different phosphorylated forms of the RNA polymerase II (RNA Pol II) C-terminal domain (CTD). We found that IR leads to global transcriptional repression of protein-coding genes, accompanied by an increase in antisense transcripts near promoters, called PROMPTs, transcribed by RNA Pol II phosphorylated on tyrosine 1 (Y1P) residue of the CTD. These Y1P-transcribed PROMPTs are enriched for PRC2 binding sites and associated with RNA Pol II proximal promoter pausing. We show the interaction between Y1P RNA Pol II and PRC2, as well as PRC2 binding to PROMPTs. Inhibition of PROMPTs or depletion of PRC2 leads to loss of transcriptional repression. Our results reveal a novel function of Y1P-dependent PROMPTs in mediating PRC2 recruitment to chromatin and RNA Pol II promoter pausing in response to DNA damage.
    DOI:  https://doi.org/10.1101/gr.278644.123
  19. J Clin Invest. 2024 Mar 14. pii: e178251. [Epub ahead of print]
    Adult and neonatal models of chemogenetic heart failure caused by oxidative stress.
    Fotios Spyropoulos, Apabrita Ayan Das, Markus Waldeck-Weiermair, Shambhu Yadav, Arvind K Pandey, Ruby Guo, Taylor A Covington, Venkata Thulabandu, Kosmas Kosmas, Benjamin Steinhorn, Mark Perella, Xiaoli Liu, Helen Christou, Thomas Michel.
      
    Keywords:  Cardiology; Heart failure; Mouse models
    DOI:  https://doi.org/10.1172/JCI178251
  20. Nat Aging. 2024 Mar 12.
    Age-dependent loss of HAPLN1 erodes vascular integrity via indirect upregulation of endothelial ICAM1 in melanoma.
    Gloria E Marino-Bravante, Alexis E Carey, Laura Hüser, Agrani Dixit, Vania Wang, Amanpreet Kaur, Ying Liu, Supeng Ding, Rahel Schnellmann, Sharon Gerecht, Luo Gu, T S Karin Eisinger-Mathason, Yash Chhabra, Ashani T Weeraratna.
      Melanoma, the most lethal form of skin cancer, often has worse outcomes in older patients. We previously demonstrated that an age-related decrease in the secreted extracellular matrix (ECM) protein HAPLN1 has a role in slowing melanoma progression. Here we show that HAPLN1 in the dermal ECM is sufficient to maintain the integrity of melanoma-associated blood vessels, as indicated by increased collagen and VE-cadherin expression. Specifically, we show that HAPLN1 in the ECM increases hyaluronic acid and decreases endothelial cell expression of ICAM1. ICAM1 phosphorylates and internalizes VE-cadherin, a critical determinant of vascular integrity, resulting in permeable blood vessels. We found that blocking ICAM1 reduces tumor size and metastasis in older mice. These results suggest that HAPLN1 alters endothelial ICAM1expression in an indirect, matrix-dependent manner. Targeting ICAM1 could be a potential treatment strategy for older patients with melanoma, emphasizing the role of aging in tumorigenesis.
    DOI:  https://doi.org/10.1038/s43587-024-00581-8
  21. Stem Cell Res Ther. 2024 Mar 13. 15(1): 76
    Short cell cycle duration is a phenotype of human epidermal stem cells.
    Tong Xiao, Ugomma C Eze, Alex Charruyer-Reinwald, Tracy Weisenberger, Ayman Khalifa, Brook Abegaze, Gabrielle K Schwab, Rasha H Elsabagh, T Richard Parenteau, Karl Kochanowski, Merisa Piper, Yumin Xia, Jeffrey B Cheng, Raymond J Cho, Ruby Ghadially.
       BACKGROUND: A traditional view is that stem cells (SCs) divide slowly. Meanwhile, both embryonic and pluripotent SCs display a shorter cell cycle duration (CCD) in comparison to more committed progenitors (CPs).
    METHODS: We examined the in vitro proliferation and cycling behavior of somatic adult human cells using live cell imaging of passage zero keratinocytes and single-cell RNA sequencing.
    RESULTS: We found two populations of keratinocytes: those with short CCD and protracted near exponential growth, and those with long CCD and terminal differentiation. Applying the ergodic principle, the comparative numbers of cycling cells in S phase in an enriched population of SCs confirmed a shorter CCD than CPs. Further, analysis of single-cell RNA sequencing of cycling adult human keratinocyte SCs and CPs indicated a shortening of both G1 and G2M phases in the SC.
    CONCLUSIONS: Contrary to the pervasive paradigm, SCs progress through cell cycle more quickly than more differentiated dividing CPs. Thus, somatic human adult keratinocyte SCs may divide infrequently, but divide rapidly when they divide. Additionally, it was found that SC-like proliferation persisted in vitro.
    Keywords:  Cell cycle duration; Cell cycle progression; Cell tracking; Differentiation; Keratinocyte; Live cell imaging; Progenitor; Single-cell RNA sequencing; Stem cell; Time-lapse microscopy
    DOI:  https://doi.org/10.1186/s13287-024-03670-y
  22. Nat Commun. 2024 Mar 12. 15(1): 2210
    ZNF827 is a single-stranded DNA binding protein that regulates the ATR-CHK1 DNA damage response pathway.
    Sile F Yang, Christopher B Nelson, Jadon K Wells, Madushan Fernando, Robert Lu, Joshua A M Allen, Lisa Malloy, Noa Lamm, Vincent J Murphy, Joel P Mackay, Andrew J Deans, Anthony J Cesare, Alexander P Sobinoff, Hilda A Pickett.
      The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway.
    DOI:  https://doi.org/10.1038/s41467-024-46578-0
  23. Nat Neurosci. 2024 Mar 12.
    Liprin-α proteins are master regulators of human presynapse assembly.
    Berta Marcó de la Cruz, Joaquín Campos, Angela Molinaro, Xingqiao Xie, Gaowei Jin, Zhiyi Wei, Claudio Acuna, Fredrik H Sterky.
      The formation of mammalian synapses entails the precise alignment of presynaptic release sites with postsynaptic receptors but how nascent cell-cell contacts translate into assembly of presynaptic specializations remains unclear. Guided by pioneering work in invertebrates, we hypothesized that in mammalian synapses, liprin-α proteins directly link trans-synaptic initial contacts to downstream steps. Here we show that, in human neurons lacking all four liprin-α isoforms, nascent synaptic contacts are formed but recruitment of active zone components and accumulation of synaptic vesicles is blocked, resulting in 'empty' boutons and loss of synaptic transmission. Interactions with presynaptic cell adhesion molecules of either the LAR-RPTP family or neurexins via CASK are required to localize liprin-α to nascent synaptic sites. Liprin-α subsequently recruits presynaptic components via a direct interaction with ELKS proteins. Thus, assembly of human presynaptic terminals is governed by a hierarchical sequence of events in which the recruitment of liprin-α proteins by presynaptic cell adhesion molecules is a critical initial step.
    DOI:  https://doi.org/10.1038/s41593-024-01592-9
  24. Nat Genet. 2024 Mar 13.
    MYC activates transcriptional enhancers to drive cancer progression.
      
    DOI:  https://doi.org/10.1038/s41588-024-01677-y
  25. J Cell Biol. 2024 Jun 03. pii: e202401057. [Epub ahead of print]223(6):
    N-cadherin dynamically regulates pediatric glioma cell migration in complex environments.
    Dayoung Kim, James M Olson, Jonathan A Cooper.
      Pediatric high-grade gliomas are highly invasive and essentially incurable. Glioma cells migrate between neurons and glia, along axon tracts, and through extracellular matrix surrounding blood vessels and underlying the pia. Mechanisms that allow adaptation to such complex environments are poorly understood. N-cadherin is highly expressed in pediatric gliomas and associated with shorter survival. We found that intercellular homotypic N-cadherin interactions differentially regulate glioma migration according to the microenvironment, stimulating migration on cultured neurons or astrocytes but inhibiting invasion into reconstituted or astrocyte-deposited extracellular matrix. N-cadherin localizes to filamentous connections between migrating leader cells but to epithelial-like junctions between followers. Leader cells have more surface and recycling N-cadherin, increased YAP1/TAZ signaling, and increased proliferation relative to followers. YAP1/TAZ signaling is dynamically regulated as leaders and followers change position, leading to altered N-cadherin levels and organization. Together, the results suggest that pediatric glioma cells adapt to different microenvironments by regulating N-cadherin dynamics and cell-cell contacts.
    DOI:  https://doi.org/10.1083/jcb.202401057
  26. J Clin Invest. 2024 Mar 15. pii: e175158. [Epub ahead of print]134(6):
    Cytoplasmic retention of the DNA/RNA-binding protein FUS ameliorates organ fibrosis in mice.
    Manuel Chiusa, Youngmin A Lee, Ming-Zhi Zhang, Raymond C Harris, Taylor Sherrill, Volkhard Lindner, Craig R Brooks, Gang Yu, Agnes B Fogo, Charles R Flynn, Jozef Zienkiewicz, Jacek Hawiger, Roy Zent, Ambra Pozzi.
      Uncontrolled accumulation of extracellular matrix leads to tissue fibrosis and loss of organ function. We previously demonstrated in vitro that the DNA/RNA-binding protein fused in sarcoma (FUS) promotes fibrotic responses by translocating to the nucleus, where it initiates collagen gene transcription. However, it is still not known whether FUS is profibrotic in vivo and whether preventing its nuclear translocation might inhibit development of fibrosis following injury. We now demonstrate that levels of nuclear FUS are significantly increased in mouse models of kidney and liver fibrosis. To evaluate the direct role of FUS nuclear translocation in fibrosis, we used mice that carry a mutation in the FUS nuclear localization sequence (FUSR521G) and the cell-penetrating peptide CP-FUS-NLS that we previously showed inhibits FUS nuclear translocation in vitro. We provide evidence that FUSR521G mice or CP-FUS-NLS-treated mice showed reduced nuclear FUS and fibrosis following injury. Finally, differential gene expression analysis and immunohistochemistry of tissues from individuals with focal segmental glomerulosclerosis or nonalcoholic steatohepatitis revealed significant upregulation of FUS and/or collagen genes and FUS protein nuclear localization in diseased organs. These results demonstrate that injury-induced nuclear translocation of FUS contributes to fibrosis and highlight CP-FUS-NLS as a promising therapeutic option for organ fibrosis.
    Keywords:  Extracellular matrix; Growth factors; Hepatology; Integrins; Nephrology
    DOI:  https://doi.org/10.1172/JCI175158
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