bims-ginsta Biomed News
on Genome instability
Issue of 2024‒05‒12
23 papers selected by
Jinrong Hu, National University of Singapore
  1. Chromosome size-dependent polar ejection force impairs mammalian mitotic error correction.
  2. Two Septin complexes mediate actin dynamics during cell wound repair.
  3. Centromeric chromatin clearings demarcate the site of kinetochore formation.
  4. Binucleated human hepatocytes arise through late cytokinetic regression during endomitosis M phase.
  5. Cell growth and nutrient availability control the mitotic exit signaling network in budding yeast.
  6. Deregulated DNA ADP-ribosylation impairs telomere replication.
  7. An interphase actin wave promotes mitochondrial content mixing and organelle homeostasis.
  8. The intrinsic substrate specificity of the human tyrosine kinome.
  9. Improved detection of DNA replication fork-associated proteins.
  10. Genome organization around nuclear speckles drives mRNA splicing efficiency.
  11. Mitochondria regulate proliferation in adult cardiac myocytes.
  12. Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis.
  13. Cerebral organoids display dynamic clonal growth and tunable tissue replenishment.
  14. Tracing embryonic hematopoiesis guides induction of pluripotent stem cells to hematopoietic progenitors.
  15. Exercise-induced changes in myocardial glucose utilization during periods of active cardiac growth.
  16. Mapping genotypes to chromatin accessibility profiles in single cells.
  17. Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters.
  18. Tissue and cellular spatiotemporal dynamics in colon aging.
  19. Design principles to tailor Hsp104 therapeutics.
  20. Nature of epigenetic aging from a single-cell perspective.
  21. Identifying Cardiomyocyte Ploidy With Nuclear Area and Volume.
  22. Regulation of Chromatin Modifications through Coordination of Nucleus Size and Epithelial Cell Morphology Heterogeneity.
  23. Converting cell death into senescence by PARP1 inhibition improves recovery from acute oxidative injury.
  1. J Cell Biol. 2024 Aug 05. pii: e202310010. [Epub ahead of print]223(8):
    Chromosome size-dependent polar ejection force impairs mammalian mitotic error correction.
    Megan K Chong, Miquel Rosas-Salvans, Vanna Tran, Sophie Dumont.
      Accurate chromosome segregation requires sister kinetochores to biorient, attaching to opposite spindle poles. To this end, the mammalian kinetochore destabilizes incorrect attachments and stabilizes correct ones, but how it discriminates between these is not yet clear. Here, we test the model that kinetochore tension is the stabilizing cue and ask how chromosome size impacts that model. We live image PtK2 cells, with just 14 chromosomes, widely ranging in size, and find that long chromosomes align at the metaphase plate later than short chromosomes. Enriching for errors and imaging error correction live, we show that long chromosomes exhibit a specific delay in correcting attachments. Using chromokinesin overexpression and laser ablation to perturb polar ejection forces, we find that chromosome size and force on arms determine alignment order. Thus, we propose a model where increased force on long chromosomes can falsely stabilize incorrect attachments, delaying their biorientation. As such, long chromosomes may require compensatory mechanisms for correcting errors to avoid chromosomal instability.
    DOI:  https://doi.org/10.1083/jcb.202310010
  2. Cell Rep. 2024 May 09. pii: S2211-1247(24)00543-6. [Epub ahead of print]43(5): 114215
    Two Septin complexes mediate actin dynamics during cell wound repair.
    Viktor Stjepić, Mitsutoshi Nakamura, Justin Hui, Susan M Parkhurst.
      Cells have robust wound repair systems to prevent further damage or infection and to quickly restore cell cortex integrity when exposed to mechanical and chemical stress. Actomyosin ring formation and contraction at the wound edge are major events during closure of the plasma membrane and underlying cytoskeleton during cell wound repair. Here, we show that all five Drosophila Septins are required for efficient cell wound repair. Based on their different recruitment patterns and knockdown/mutant phenotypes, two distinct Septin complexes, Sep1/Sep2/Pnut and Sep4/Sep5/Pnut, are assembled to regulate actin ring assembly, contraction, and remodeling during the repair process. Intriguingly, we find that these two Septin complexes have different F-actin bending activities. In addition, we find that Anillin regulates the recruitment of only one of two Septin complexes upon wounding. Our results demonstrate that two functionally distinct Septin complexes work side by side to discretely regulate actomyosin ring dynamics during cell wound repair.
    Keywords:  Anillin; CP: Cell biology; Drosophila; Septins; actin bending; actin bundling; cell wound repair; cytoskeleton
    DOI:  https://doi.org/10.1016/j.celrep.2024.114215
  3. bioRxiv. 2024 Apr 26. pii: 2024.04.26.591177. [Epub ahead of print]
    Centromeric chromatin clearings demarcate the site of kinetochore formation.
    Kathryn Kixmoeller, Yi-Wei Chang, Ben E Black.
      The centromere is the chromosomal locus that recruits the kinetochore, directing faithful propagation of the genome during cell division. The kinetochore has been interrogated by electron microscopy since the middle of the last century, but with methodologies that compromised fine structure. Using cryo-ET on human mitotic chromosomes, we reveal a distinctive architecture at the centromere: clustered 20-25 nm nucleosome-associated complexes within chromatin clearings that delineate them from surrounding chromatin. Centromere components CENP-C and CENP-N are each required for the integrity of the complexes, while CENP-C is also required to maintain the chromatin clearing. We further visualize the scaffold of the fibrous corona, a structure amplified at unattached kinetochores, revealing crescent-shaped parallel arrays of fibrils that extend >1 μm. Thus, we reveal how the organization of centromeric chromatin creates a clearing at the site of kinetochore formation as well as the nature of kinetochore amplification mediated by corona fibrils.
    DOI:  https://doi.org/10.1101/2024.04.26.591177
  4. J Cell Biol. 2024 Aug 05. pii: e202403020. [Epub ahead of print]223(8):
    Binucleated human hepatocytes arise through late cytokinetic regression during endomitosis M phase.
    Gabriella S Darmasaputra, Cindy C Geerlings, Susana M Chuva de Sousa Lopes, Hans Clevers, Matilde Galli.
      Binucleated polyploid cells are common in many animal tissues, where they arise by endomitosis, a non-canonical cell cycle in which cells enter M phase but do not undergo cytokinesis. Different steps of cytokinesis have been shown to be inhibited during endomitosis M phase in rodents, but it is currently unknown how human cells undergo endomitosis. In this study, we use fetal-derived human hepatocyte organoids (Hep-Orgs) to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, which is associated with the loss of four cortical anchoring proteins, RacGAP1, Anillin, SEPT9, and citron kinase (CIT-K). Moreover, reduction of WNT activity increases the percentage of binucleated cells in Hep-Orgs, an effect that is dependent on the atypical E2F proteins, E2F7 and E2F8. Together, we have elucidated how hepatocytes undergo endomitosis in human Hep-Orgs, providing new insights into the mechanisms of endomitosis in mammals.
    DOI:  https://doi.org/10.1083/jcb.202403020
  5. J Cell Biol. 2024 Aug 05. pii: e202305008. [Epub ahead of print]223(8):
    Cell growth and nutrient availability control the mitotic exit signaling network in budding yeast.
    Rafael A Talavera, Beth E Prichard, Robert A Sommer, Ricardo M Leitao, Christopher J Sarabia, Semin Hazir, Joao A Paulo, Steven P Gygi, Douglas R Kellogg.
      Cell growth is required for cell cycle progression. The amount of growth required for cell cycle progression is reduced in poor nutrients, which leads to a reduction in cell size. In budding yeast, nutrients can influence cell size by modulating the extent of bud growth, which occurs predominantly in mitosis. However, the mechanisms are unknown. Here, we used mass spectrometry to identify proteins that modulate bud growth in response to nutrient availability. This led to the discovery that nutrients regulate numerous components of the mitotic exit network (MEN), which controls exit from mitosis. A key component of the MEN undergoes gradual multisite phosphorylation during bud growth that is dependent upon bud growth and correlated with the extent of growth. Furthermore, activation of the MEN is sufficient to override a growth requirement for mitotic exit. The data suggest a model in which the MEN ensures that mitotic exit occurs only when an appropriate amount of bud growth has occurred.
    DOI:  https://doi.org/10.1083/jcb.202305008
  6. Nat Struct Mol Biol. 2024 May 07.
    Deregulated DNA ADP-ribosylation impairs telomere replication.
    Anne R Wondisford, Junyeop Lee, Robert Lu, Marion Schuller, Josephine Groslambert, Ragini Bhargava, Sandra Schamus-Haynes, Leyneir C Cespedes, Patricia L Opresko, Hilda A Pickett, Jaewon Min, Ivan Ahel, Roderick J O'Sullivan.
      The recognition that DNA can be ADP ribosylated provides an unexpected regulatory level of how ADP-ribosylation contributes to genome stability, epigenetics and immunity. Yet, it remains unknown whether DNA ADP-ribosylation (DNA-ADPr) promotes genome stability and how it is regulated. Here, we show that telomeres are subject to DNA-ADPr catalyzed by PARP1 and removed by TARG1. Mechanistically, we show that DNA-ADPr is coupled to lagging telomere DNA strand synthesis, forming at single-stranded DNA present at unligated Okazaki fragments and on the 3' single-stranded telomere overhang. Persistent DNA-linked ADPr, due to TARG1 deficiency, eventually leads to telomere shortening. Furthermore, using the bacterial DNA ADP-ribosyl-transferase toxin to modify DNA at telomeres directly, we demonstrate that unhydrolyzed DNA-linked ADP-ribose compromises telomere replication and telomere integrity. Thus, by identifying telomeres as chromosomal targets of PARP1 and TARG1-regulated DNA-ADPr, whose deregulation compromises telomere replication and integrity, our study highlights and establishes the critical importance of controlling DNA-ADPr turnover for sustained genome stability.
    DOI:  https://doi.org/10.1038/s41594-024-01279-6
  7. Nat Commun. 2024 May 07. 15(1): 3793
    An interphase actin wave promotes mitochondrial content mixing and organelle homeostasis.
    Stephen M Coscia, Andrew S Moore, Cameron P Thompson, Christian F Tirrito, E Michael Ostap, Erika L F Holzbaur.
      Across the cell cycle, mitochondrial dynamics are regulated by a cycling wave of actin polymerization/depolymerization. In metaphase, this wave induces actin comet tails on mitochondria that propel these organelles to drive spatial mixing, resulting in their equitable inheritance by daughter cells. In contrast, during interphase the cycling actin wave promotes localized mitochondrial fission. Here, we identify the F-actin nucleator/elongator FMNL1 as a positive regulator of the wave. FMNL1-depleted cells exhibit decreased mitochondrial polarization, decreased mitochondrial oxygen consumption, and increased production of reactive oxygen species. Accompanying these changes is a loss of hetero-fusion of wave-fragmented mitochondria. Thus, we propose that the interphase actin wave maintains mitochondrial homeostasis by promoting mitochondrial content mixing. Finally, we investigate the mechanistic basis for the observation that the wave drives mitochondrial motility in metaphase but mitochondrial fission in interphase. Our data indicate that when the force of actin polymerization is resisted by mitochondrial tethering to microtubules, as in interphase, fission results.
    DOI:  https://doi.org/10.1038/s41467-024-48189-1
  8. Nature. 2024 May 08.
    The intrinsic substrate specificity of the human tyrosine kinome.
    Tomer M Yaron-Barir, Brian A Joughin, Emily M Huntsman, Alexander Kerelsky, Daniel M Cizin, Benjamin M Cohen, Amit Regev, Junho Song, Neil Vasan, Ting-Yu Lin, Jose M Orozco, Christina Schoenherr, Cari Sagum, Mark T Bedford, R Max Wynn, Shih-Chia Tso, David T Chuang, Lei Li, Shawn S-C Li, Pau Creixell, Konstantin Krismer, Mina Takegami, Harin Lee, Bin Zhang, Jingyi Lu, Ian Cossentino, Sean D Landry, Mohamed Uduman, John Blenis, Olivier Elemento, Margaret C Frame, Peter V Hornbeck, Lewis C Cantley, Benjamin E Turk, Michael B Yaffe, Jared L Johnson.
      Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.
    DOI:  https://doi.org/10.1038/s41586-024-07407-y
  9. Cell Rep. 2024 May 02. pii: S2211-1247(24)00506-0. [Epub ahead of print]43(5): 114178
    Improved detection of DNA replication fork-associated proteins.
    Rebecca S Rivard, Ya-Chu Chang, Ryan L Ragland, Yee-Mon Thu, Muzaffer Kassab, Rahul Shubhra Mandal, Susan K Van Riper, Katarzyna Kulej, LeeAnn Higgins, Todd M Markowski, David Shang, Jack Hedberg, Luke Erber, Benjamin Garcia, Yue Chen, Anja-Katrin Bielinsky, Eric J Brown.
      Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls, and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of diglycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components and nuclear cytoskeleton networks.
    Keywords:  CP: Molecular biology; DNA repair; DNA replication; DRIPPER; iPOND; nuclear pore complex; p97; replication stress; replisome; ubiquination
    DOI:  https://doi.org/10.1016/j.celrep.2024.114178
  10. Nature. 2024 May 08.
    Genome organization around nuclear speckles drives mRNA splicing efficiency.
    Prashant Bhat, Amy Chow, Benjamin Emert, Olivia Ettlin, Sofia A Quinodoz, Mackenzie Strehle, Yodai Takei, Alex Burr, Isabel N Goronzy, Allen W Chen, Wesley Huang, Jose Lorenzo M Ferrer, Elizabeth Soehalim, Say-Tar Goh, Tara Chari, Delaney K Sullivan, Mario R Blanco, Mitchell Guttman.
      The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.
    DOI:  https://doi.org/10.1038/s41586-024-07429-6
  11. J Clin Invest. 2024 May 09. pii: e165482. [Epub ahead of print]
    Mitochondria regulate proliferation in adult cardiac myocytes.
    Gregory B Waypa, Kimberly A Smith, Paul T Mungai, Vincent J Dudley, Kathryn A Helmin, Benjamin D Singer, Clara Bien Peek, Joseph Bass, Lauren Beussink-Nelson, Sanjiv J Shah, Gaston Ofman, J Andrew Wasserstrom, William A Muller, Alexander V Misharin, G R Scott Budinger, Hiam Abdala-Valencia, Navdeep S Chandel, Danijela Dokic, Elizabeth T Bartom, Shuang Zhang, Yuki Tatekoshi, Amir Mahmoodzadeh, Hossein Ardehali, Edward B Thorp, Paul T Schumacker.
      Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity. We tested whether forced reversal of adult cardiomyocytes back to a fetal glycolytic phenotype would restore proliferative capacity. We deleted Uqcrfs1 (mitochondrial Rieske Iron-Sulfur protein, RISP) in hearts of adult mice. As RISP protein decreased, heart mitochondrial function declined, and glucose utilization increased. Simultaneously, they underwent hyperplastic remodeling during which cardiomyocyte number doubled without cellular hypertrophy. Cellular energy supply was preserved, AMPK activation was absent, and mTOR activation was evident. In ischemic hearts with RISP deletion, new cardiomyocytes migrated into the infarcted region, suggesting the potential for therapeutic cardiac regeneration. RNA-seq revealed upregulation of genes associated with cardiac development and proliferation. Metabolomic analysis revealed a decrease in alpha-ketoglutarate (required for TET-mediated demethylation) and an increase in S-adenosylmethionine (required for methyltransferase activity). Analysis revealed an increase in methylated CpGs near gene transcriptional start sites. Genes that were both differentially expressed and differentially methylated were linked to upregulated cardiac developmental pathways. We conclude that decreased mitochondrial function and increased glucose utilization can restore mitotic capacity in adult cardiomyocytes resulting in the generation of new heart cells, potentially through the modification of substrates that regulate epigenetic modification of genes required for proliferation.
    Keywords:  Bioenergetics; Cardiology; Cardiovascular disease; Metabolism; Mitochondria
    DOI:  https://doi.org/10.1172/JCI165482
  12. Circulation. 2024 May 06.
    Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis.
    Feng Xiao, Ngoc Uyen Nhi Nguyen, Ping Wang, Shujuan Li, Ching-Cheng Hsu, Suwannee Thet, Wataru Kimura, Xiang Luo, Nicholas T Lam, Ivan Menendez-Montes, Waleed Elhelaly, Alisson Campos Cardoso, Ana Helena Macedo Pereira, Rohit Singh, Sakthivel Sadayappan, Mohammed Kanchwala, Chao Xing, Feria A Ladha, J Travis Hinson, Roger J Hajjar, Joseph A Hill, Hesham A Sadek.
      BACKGROUND: Recent interest in understanding cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow the abscission of daughter cardiomyocytes.METHODS: Here, we use a proteomics screen to identify adducin, an actin capping protein previously not studied in cardiomyocytes, as a regulator of sarcomere disassembly. We generated many adeno-associated viruses and cardiomyocyte-specific genetic gain-of-function models to examine the role of adducin in neonatal and adult cardiomyocytes in vitro and in vivo.
    RESULTS: We identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phospho-isoforms of α-adducin in identified Thr445/Thr480 phosphorylation of a short isoform of α-adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this α-adducin variant along with γ-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with α-actinin.
    CONCLUSIONS: These results highlight an important mechanism for coordinating cytoskeletal morphological changes during cardiomyocyte mitosis.
    Keywords:  Irak4; adducin; cardiac proliferation; sarcomere disassembly; α-actinin
    DOI:  https://doi.org/10.1161/CIRCULATIONAHA.122.059102
  13. Nat Cell Biol. 2024 May 07.
    Cerebral organoids display dynamic clonal growth and tunable tissue replenishment.
    Dominik Lindenhofer, Simon Haendeler, Christopher Esk, Jamie B Littleboy, Clarisse Brunet Avalos, Julia Naas, Florian G Pflug, Eline G P van de Ven, Daniel Reumann, Alexandre D Baffet, Arndt von Haeseler, Jürgen A Knoblich.
      During brain development, neural progenitors expand through symmetric divisions before giving rise to differentiating cell types via asymmetric divisions. Transition between those modes varies among individual neural stem cells, resulting in clones of different sizes. Imaging-based lineage tracing allows for lineage analysis at high cellular resolution but systematic approaches to analyse clonal behaviour of entire tissues are currently lacking. Here we implement whole-tissue lineage tracing by genomic DNA barcoding in 3D human cerebral organoids, to show that individual stem cell clones produce progeny on a vastly variable scale. By using stochastic modelling we find that variable lineage sizes arise because a subpopulation of lineages retains symmetrically dividing cells. We show that lineage sizes can adjust to tissue demands after growth perturbation via chemical ablation or genetic restriction of a subset of cells in chimeric organoids. Our data suggest that adaptive plasticity of stem cell populations ensures robustness of development in human brain organoids.
    DOI:  https://doi.org/10.1038/s41556-024-01412-z
  14. Dev Cell. 2024 May 06. pii: S1534-5807(24)00226-0. [Epub ahead of print]59(9): 1093-1095
    Tracing embryonic hematopoiesis guides induction of pluripotent stem cells to hematopoietic progenitors.
    Xinjian Mao, Linheng Li.
      In this issue of Developmental Cell, Fowler et al. applied genetic lineage-tracing mouse models to support the notion that artery endothelial cells are the predominant source of hematopoietic stem cells. They leveraged this and developed a method capable of efficiently differentiating human pluripotent stem cells into HLF+HOXA+ hematopoietic progenitors.
    DOI:  https://doi.org/10.1016/j.devcel.2024.04.001
  15. J Mol Cell Cardiol. 2024 May 03. pii: S0022-2828(24)00064-6. [Epub ahead of print]191 50-62
    Exercise-induced changes in myocardial glucose utilization during periods of active cardiac growth.
    Kyle L Fulghum, Helen E Collins, Pawel K Lorkiewicz, Teresa A Cassel, Teresa W M Fan, Bradford G Hill.
      Exercise training can promote physiological cardiac growth, which has been suggested to involve changes in glucose metabolism to facilitate hypertrophy of cardiomyocytes. In this study, we used a dietary, in vivo isotope labeling approach to examine how exercise training influences the metabolic fate of carbon derived from dietary glucose in the heart during acute, active, and established phases of exercise-induced cardiac growth. Male and female FVB/NJ mice were subjected to treadmill running for up to 4 weeks and cardiac growth was assessed by gravimetry. Cardiac metabolic responses to exercise were assessed via in vivo tracing of [13C6]-glucose via mass spectrometry and nuclear magnetic resonance. We found that the half-maximal cardiac growth response was achieved by approximately 1 week of daily exercise training, with near maximal growth observed in male mice with 2 weeks of training; however, female mice were recalcitrant to exercise-induced cardiac growth and required a higher daily intensity of exercise training to achieve significant, albeit modest, increases in cardiac mass. We also found that increases in the energy charge of adenylate and guanylate nucleotide pools precede exercise-induced changes in cardiac size and were associated with higher glucose tracer enrichment in the TCA pool and in amino acids (aspartate, glutamate) sourced by TCA intermediates. Our data also indicate that the activity of collateral biosynthetic pathways of glucose metabolism may not be markedly altered by exercise. Overall, this study provides evidence that metabolic remodeling in the form of heightened energy charge and increased TCA cycle activity and cataplerosis precedes cardiac growth caused by exercise training in male mice.
    DOI:  https://doi.org/10.1016/j.yjmcc.2024.04.014
  16. Nature. 2024 May 08.
    Mapping genotypes to chromatin accessibility profiles in single cells.
    Franco Izzo, Robert M Myers, Saravanan Ganesan, Levan Mekerishvili, Sanjay Kottapalli, Tamara Prieto, Elliot O Eton, Theo Botella, Andrew J Dunbar, Robert L Bowman, Jesus Sotelo, Catherine Potenski, Eleni P Mimitou, Maximilian Stahl, Sebastian El Ghaity-Beckley, JoAnn Arandela, Ramya Raviram, Daniel C Choi, Ronald Hoffman, Ronan Chaligné, Omar Abdel-Wahab, Peter Smibert, Irene M Ghobrial, Joseph M Scandura, Bridget Marcellino, Ross L Levine, Dan A Landau.
      In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.
    DOI:  https://doi.org/10.1038/s41586-024-07388-y
  17. Nat Methods. 2024 May 09.
    Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters.
    Jean-Benoît Lalanne, Samuel G Regalado, Silvia Domcke, Diego Calderon, Beth K Martin, Xiaoyi Li, Tony Li, Chase C Suiter, Choli Lee, Cole Trapnell, Jay Shendure.
      The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.
    DOI:  https://doi.org/10.1038/s41592-024-02260-3
  18. bioRxiv. 2024 Apr 26. pii: 2024.04.22.590125. [Epub ahead of print]
    Tissue and cellular spatiotemporal dynamics in colon aging.
    Aidan C Daly, Francesco Cambuli, Tarmo Äijö, Britta Lötstedt, Nemanja Marjanovic, Olena Kuksenko, Matthew Smith-Erb, Sara Fernandez, Daniel Domovic, Nicholas Van Wittenberghe, Eugene Drokhlyansky, Gabriel K Griffin, Hemali Phatnani, Richard Bonneau, Aviv Regev, Sanja Vickovic.
      Tissue structure and molecular circuitry in the colon can be profoundly impacted by systemic age-related effects, but many of the underlying molecular cues remain unclear. Here, we built a cellular and spatial atlas of the colon across three anatomical regions and 11 age groups, encompassing ∼1,500 mouse gut tissues profiled by spatial transcriptomics and ∼400,000 single nucleus RNA-seq profiles. We developed a new computational framework, cSplotch, which learns a hierarchical Bayesian model of spatially resolved cellular expression associated with age, tissue region, and sex, by leveraging histological features to share information across tissue samples and data modalities. Using this model, we identified cellular and molecular gradients along the adult colonic tract and across the main crypt axis, and multicellular programs associated with aging in the large intestine. Our multi-modal framework for the investigation of cell and tissue organization can aid in the understanding of cellular roles in tissue-level pathology.
    DOI:  https://doi.org/10.1101/2024.04.22.590125
  19. bioRxiv. 2024 Apr 28. pii: 2024.04.26.591398. [Epub ahead of print]
    Design principles to tailor Hsp104 therapeutics.
    JiaBei Lin, Peter J Carman, Craig W Gambogi, Nathan M Kendsersky, Edward Chuang, Stephanie N Gates, Adam L Yokom, Alexandrea N Rizo, Daniel R Southworth, James Shorter.
      The hexameric AAA+ disaggregase, Hsp104, collaborates with Hsp70 and Hsp40 via its autoregulatory middle domain (MD) to solubilize aggregated protein conformers. However, how ATP- or ADP-specific MD configurations regulate Hsp104 hexamers remains poorly understood. Here, we define an ATP-specific network of interprotomer contacts between nucleotide-binding domain 1 (NBD1) and MD helix L1, which tunes Hsp70 collaboration. Manipulating this network can: (a) reduce Hsp70 collaboration without enhancing activity; (b) generate Hsp104 hypomorphs that collaborate selectively with class B Hsp40s; (c) produce Hsp70-independent potentiated variants; or (d) create species barriers between Hsp104 and Hsp70. Conversely, ADP-specific intraprotomer contacts between MD helix L2 and NBD1 restrict activity, and their perturbation frequently potentiates Hsp104. Importantly, adjusting the NBD1:MD helix L1 rheostat via rational design enables finely tuned collaboration with Hsp70 to safely potentiate Hsp104, minimize off- target toxicity, and counteract FUS proteinopathy in human cells. Thus, we establish important design principles to tailor Hsp104 therapeutics.
    DOI:  https://doi.org/10.1101/2024.04.26.591398
  20. Nat Aging. 2024 May 09.
    Nature of epigenetic aging from a single-cell perspective.
    Andrei E Tarkhov, Thomas Lindstrom-Vautrin, Sirui Zhang, Kejun Ying, Mahdi Moqri, Bohan Zhang, Alexander Tyshkovskiy, Orr Levy, Vadim N Gladyshev.
      Age-related changes in DNA methylation (DNAm) form the basis of the most robust predictors of age-epigenetic clocks-but a clear mechanistic understanding of exactly which aspects of aging are quantified by these clocks is lacking. Here, to clarify the nature of epigenetic aging, we juxtapose the dynamics of tissue and single-cell DNAm in mice. We compare these changes during early development with those observed during adult aging in mice, and corroborate our analyses with a single-cell RNA sequencing analysis within the same multiomics dataset. We show that epigenetic aging involves co-regulated changes as well as a major stochastic component, and this is consistent with transcriptional patterns. We further support the finding of stochastic epigenetic aging by direct tissue and single-cell DNAm analyses and modeling of aging DNAm trajectories with a stochastic process akin to radiocarbon decay. Finally, we describe a single-cell algorithm for the identification of co-regulated and stochastic CpG clusters showing consistent transcriptomic coordination patterns. Together, our analyses increase our understanding of the basis of epigenetic clocks and highlight potential opportunities for targeting aging and evaluating longevity interventions.
    DOI:  https://doi.org/10.1038/s43587-024-00616-0
  21. Circulation. 2024 May 07. 149(19): 1540-1542
    Identifying Cardiomyocyte Ploidy With Nuclear Area and Volume.
    Zehao Yao, Lina Bai, Kefei Dou, Yu Nie.
      
    Keywords:  cardiomegaly; cell nucleus size; in situ hybridization, fluorescence; myocytes, cardiac; ploidies
    DOI:  https://doi.org/10.1161/CIRCULATIONAHA.123.065507
  22. bioRxiv. 2024 Apr 22. pii: 2024.04.18.590164. [Epub ahead of print]
    Regulation of Chromatin Modifications through Coordination of Nucleus Size and Epithelial Cell Morphology Heterogeneity.
    Alexandra Bermudez, Zoe D Latham, Alex J Ma, Dapeng Bi, Jimmy K Hu, Neil Y C Lin.
      Cell morphology heterogeneity within epithelial collectives is a pervasive phenomenon intertwined with tissue mechanical properties. Despite its widespread occurrence, the underlying mechanisms driving cell morphology heterogeneity and its consequential biological ramifications remain elusive. Here, we investigate the dynamic evolution of epithelial cell morphology and nucleus morphology during crowding, unveiling a consistent correlation between the two. Our investigation reveals a persistent log-normal probability distribution characterizing both cell and nucleus areas across diverse crowding stages and epithelial model systems. We showed that this morphological diversity arises from asymmetric partitioning during cell division and is perpetuated through actomyosin-mediated regulation of cell-nucleus size coordination. Moreover, we provide insights into the impact of nucleus morphology on chromatin dynamics, demonstrating that constraining nucleus area leads to downregulation of the euchromatic mark H3K9ac and upregulation of the heterochromatic mark H3K27me3 through modulation of histone demethylase UTX expression. These findings under-score the significance of cell morphology heterogeneity as a driver of chromatin state diversity, shaping functional variability within epithelial tissues.
    DOI:  https://doi.org/10.1101/2024.04.18.590164
  23. Nat Aging. 2024 May 09.
    Converting cell death into senescence by PARP1 inhibition improves recovery from acute oxidative injury.
    Jamil Nehme, Lina Mesilmany, Marta Varela-Eirin, Simone Brandenburg, Abdullah Altulea, Yao Lin, Mariana Gaya da Costa, Marc Seelen, Jan-Luuk Hillebrands, Harry van Goor, Raya Saab, Haidar Akl, Natacha Prevarskaya, Valerio Farfariello, Marco Demaria.
      Excessive amounts of reactive oxygen species (ROS) lead to macromolecular damage and high levels of cell death with consequent pathological sequelae. We hypothesized that switching cell death to a tissue regenerative state could potentially improve the short-term and long-term detrimental effects of ROS-associated acute tissue injury, although the mechanisms regulating oxidative stress-induced cell fate decisions and their manipulation for improving repair are poorly understood. Here, we show that cells exposed to high oxidative stress enter a poly (ADP-ribose) polymerase 1 (PARP1)-mediated regulated cell death, and that blocking PARP1 activation promotes conversion of cell death into senescence (CODIS). We demonstrate that this conversion depends on reducing mitochondrial Ca2+ overload as a consequence of retaining the hexokinase II on mitochondria. In a mouse model of kidney ischemia-reperfusion damage, PARP inhibition reduces necrosis and increases transient senescence at the injury site, alongside improved recovery from damage. Together, these data provide evidence that converting cell death into transient senescence can therapeutically benefit tissue regeneration.
    DOI:  https://doi.org/10.1038/s43587-024-00627-x
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