bims-ginsta Biomed News
on Genome instability
Issue of 2024–03–10
27 papers selected by
Jinrong Hu, National University of Singapore
  1. Safeguarding the epigenome through the cell cycle: a multitasking game.
  2. Structure of the human outer kinetochore KMN network complex.
  3. A surge in cytoplasmic viscosity triggers nuclear remodeling required for Dux silencing and pre-implantation embryo development.
  4. RNAP II antagonizes mitotic chromatin folding and chromosome segregation by condensin.
  5. Decoding chromatin states by proteomic profiling of nucleosome readers.
  6. Regulation of transcription patterns, poly(ADP-ribose), and RNA-DNA hybrids by the ATM protein kinase.
  7. Cytokinetic abscission requires actin-dependent microtubule severing.
  8. MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.
  9. Glycerophosphodiesters inhibit lysosomal phospholipid catabolism in Batten disease.
  10. RTF2 controls replication repriming and ribonucleotide excision at the replisome.
  11. Sister chromatid cohesion halts DNA loop expansion.
  12. Microtubule damage shapes the acetylation gradient.
  13. A SWI/SNF-dependent transcriptional regulation mediated by POU2AF2/C11orf53 at enhancer.
  14. Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength.
  15. DUX4-induced HSATII transcription causes KDM2A/B-PRC1 nuclear foci and impairs DNA damage response.
  16. Structure of the human KMN complex and implications for regulation of its assembly.
  17. Integrated stress response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation.
  18. Tissue mosaicism following stem cell aging: blood as an exemplar.
  19. Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle.
  20. In-cell NMR suggests that DNA i-motif levels are strongly depleted in living human cells.
  21. A mitochondrial NADPH-cholesterol axis regulates extracellular vesicle biogenesis to support hematopoietic stem cell fate.
  22. An in vivo screening platform identifies senolytic compounds that target p16INK4a+ fibroblasts in lung fibrosis.
  23. A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes, as well as failure of epigenetic reprogramming and zygotic genome activation in embryos.
  24. Distinct pathways drive anterior hypoblast specification in the implanting human embryo.
  25. Synthetic reversed sequences reveal default genomic states.
  26. GRB2 stabilizes RAD51 at reversed replication forks suppressing genomic instability and innate immunity against cancer.
  27. scAbsolute: measuring single-cell ploidy and replication status.
  1. Curr Opin Genet Dev. 2024 Mar 05. pii: S0959-437X(24)00010-8. [Epub ahead of print]85 102161
    Safeguarding the epigenome through the cell cycle: a multitasking game.
    Valentin Flury, Anja Groth.
      Sustaining cell identity and function across cell division is germane to human development, healthspan, and cancer avoidance. This relies significantly on propagation of chromatin organization between cell generations, as chromatin presents a barrier to cell fate and cell state conversions. Inheritance of chromatin states across the many cell divisions required for development and tissue homeostasis represents a major challenge, especially because chromatin is disrupted to allow passage of the DNA replication fork to synthesize the two daughter strands. This process also leads to a twofold dilution of epigenetic information in histones, which needs to be accurately restored for faithful propagation of chromatin states across cell divisions. Recent research has identified distinct multilayered mechanisms acting to propagate epigenetic information to daughter strands. Here, we summarize key principles of how epigenetic information in parental histones is transferred across DNA replication and how new histones robustly acquire the same information postreplication, representing a core component of epigenetic cell memory.
    DOI:  https://doi.org/10.1016/j.gde.2024.102161
  2. Nat Struct Mol Biol. 2024 Mar 08.
    Structure of the human outer kinetochore KMN network complex.
    Stanislau Yatskevich, Jing Yang, Dom Bellini, Ziguo Zhang, David Barford.
      Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network that comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C, augmented by multiple peptidic inter-subunit connections. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our study indicates how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation might restrict outer kinetochore assembly to functional centromeres during cell division.
    DOI:  https://doi.org/10.1038/s41594-024-01249-y
  3. Cell Rep. 2024 Mar 05. pii: S2211-1247(24)00245-6. [Epub ahead of print]43(3): 113917
    A surge in cytoplasmic viscosity triggers nuclear remodeling required for Dux silencing and pre-implantation embryo development.
    Yunan Ye, Hayden Anthony Homer.
      Embryonic genome activation (EGA) marks the transition from dependence on maternal transcripts to an embryonic transcriptional program. The precise temporal regulation of gene expression, specifically the silencing of the Dux/murine endogenous retrovirus type L (MERVL) program during late 2-cell interphase, is crucial for developmental progression in mouse embryos. How this finely tuned regulation is achieved within this specific window is poorly understood. Here, using particle-tracking microrheology throughout the mouse oocyte-to-embryo transition, we identify a surge in cytoplasmic viscosity specific to late 2-cell interphase brought about by high microtubule and endomembrane density. Importantly, preventing the rise in 2-cell viscosity severely impairs nuclear reorganization, resulting in a persistently open chromatin configuration and failure to silence Dux/MERVL. This, in turn, derails embryo development beyond the 2- and 4-cell stages. Our findings reveal a mechanical role of the cytoplasm in regulating Dux/MERVL repression via nuclear remodeling during a temporally confined period in late 2-cell interphase.
    Keywords:  2-cell embryo; CP: Developmental biology; CP: Molecular biology; Dux; Heterochromatin; MERVL; chromocenter; cytoplasmic viscosity; embryonic genome activation; microrheology; oocyte; preimplantation development
    DOI:  https://doi.org/10.1016/j.celrep.2024.113917
  4. Cell Rep. 2024 Mar 05. pii: S2211-1247(24)00229-8. [Epub ahead of print]43(3): 113901
    RNAP II antagonizes mitotic chromatin folding and chromosome segregation by condensin.
    Jérémy Lebreton, Léonard Colin, Elodie Chatre, Pascal Bernard.
      Condensin shapes mitotic chromosomes by folding chromatin into loops, but whether it does so by DNA-loop extrusion remains speculative. Although loop-extruding cohesin is stalled by transcription, the impact of transcription on condensin, which is enriched at highly expressed genes in many species, remains unclear. Using degrons of Rpb1 or the torpedo nuclease Dhp1XRN2 to either deplete or displace RNAPII on chromatin in fission yeast metaphase cells, we show that RNAPII does not load condensin on DNA. Instead, RNAPII retains condensin in cis and hinders its ability to fold mitotic chromatin and to support chromosome segregation, consistent with the stalling of a loop extruder. Transcription termination by Dhp1 limits such a hindrance. Our results shed light on the integrated functioning of condensin, and we argue that a tight control of transcription underlies mitotic chromosome assembly by loop-extruding condensin.
    Keywords:  CP: Molecular biology; SMC complexes; condensin; loop extrusion; mitotic chromosome assembly; transcription; transcription-termination
    DOI:  https://doi.org/10.1016/j.celrep.2024.113901
  5. Nature. 2024 Mar 06.
    Decoding chromatin states by proteomic profiling of nucleosome readers.
    Saulius Lukauskas, Andrey Tvardovskiy, Nhuong V Nguyen, Mara Stadler, Peter Faull, Tina Ravnsborg, Bihter Özdemir Aygenli, Scarlett Dornauer, Helen Flynn, Rik G H Lindeboom, Teresa K Barth, Kevin Brockers, Stefanie M Hauck, Michiel Vermeulen, Ambrosius P Snijders, Christian L Müller, Peter A DiMaggio, Ole N Jensen, Robert Schneider, Till Bartke.
      DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome1,2. While many 'readers' of individual modifications have been described3-5, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins with over 80 modified dinucleosomes representing promoter, enhancer and heterochromatin states. By deconvoluting complex nucleosome-binding profiles into networks of co-regulated proteins and distinct nucleosomal features driving protein recruitment or exclusion, we show comprehensively how chromatin states are decoded by chromatin readers. We find highly distinctive binding responses to different features, many factors that recognize multiple features, and that nucleosomal modifications and linker DNA operate largely independently in regulating protein binding to chromatin. Our online resource, the Modification Atlas of Regulation by Chromatin States (MARCS), provides in-depth analysis tools to engage with our results and advance the discovery of fundamental principles of genome regulation by chromatin states.
    DOI:  https://doi.org/10.1038/s41586-024-07141-5
  6. Cell Rep. 2024 Mar 04. pii: S2211-1247(24)00224-9. [Epub ahead of print]43(3): 113896
    Regulation of transcription patterns, poly(ADP-ribose), and RNA-DNA hybrids by the ATM protein kinase.
    Phillip R Woolley, Xuemei Wen, Olivia M Conway, Nicolette A Ender, Ji-Hoon Lee, Tanya T Paull.
      The ataxia telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response and also an important sensor of oxidative stress. Analysis of gene expression in ataxia-telangiectasia (A-T) patient brain tissue shows that large-scale transcriptional changes occur in patient cerebellum that correlate with the expression level and guanine-cytosine (GC) content of transcribed genes. In human neuron-like cells in culture, we map locations of poly(ADP-ribose) and RNA-DNA hybrid accumulation genome-wide with ATM inhibition and find that these marks also coincide with high transcription levels, active transcription histone marks, and high GC content. Antioxidant treatment reverses the accumulation of R-loops in transcribed regions, consistent with the central role of reactive oxygen species in promoting these lesions. Based on these results, we postulate that transcription-associated lesions accumulate in ATM-deficient cells and that the single-strand breaks and PARylation at these sites ultimately generate changes in transcription that compromise cerebellum function and lead to neurodegeneration over time in A-T patients.
    Keywords:  ATM; CP: Molecular biology; DNA repair; R-loops; cerebellar ataxia; poly-ADP-ribosylation; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2024.113896
  7. Nat Commun. 2024 Mar 02. 15(1): 1949
    Cytokinetic abscission requires actin-dependent microtubule severing.
    Tamara Advedissian, Stéphane Frémont, Arnaud Echard.
      Cell division is completed by the abscission of the intercellular bridge connecting the daughter cells. Abscission requires the polymerization of an ESCRT-III cone close to the midbody to both recruit the microtubule severing enzyme spastin and scission the plasma membrane. Here, we found that the microtubule and the membrane cuts are two separate events that are regulated differently. Using HeLa cells, we uncovered that the F-actin disassembling protein Cofilin-1 controls the disappearance of a transient pool of branched F-actin which is precisely assembled at the tip of the ESCRT-III cone shortly before the microtubule cut. Functionally, Cofilin-1 and Arp2/3-mediated branched F-actin favor abscission by promoting local severing of the microtubules but do not participate later in the membrane scission event. Mechanistically, we propose that branched F-actin functions as a physical barrier that limits ESCRT-III cone elongation and thereby favors stable spastin recruitment. Our work thus reveals that F-actin controls the timely and local disassembly of microtubules required for cytokinetic abscission.
    DOI:  https://doi.org/10.1038/s41467-024-46062-9
  8. Nat Genet. 2024 Mar 07.
    MYC activity at enhancers drives prognostic transcriptional programs through an epigenetic switch.
    Simon T Jakobsen, Rikke A M Jensen, Maria S Madsen, Tina Ravnsborg, Christian S Vaagenso, Majken S Siersbæk, Hjorleifur Einarsson, Robin Andersson, Ole N Jensen, Rasmus Siersbæk.
      The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.
    DOI:  https://doi.org/10.1038/s41588-024-01676-z
  9. Mol Cell. 2024 Feb 28. pii: S1097-2765(24)00125-4. [Epub ahead of print]
    Glycerophosphodiesters inhibit lysosomal phospholipid catabolism in Batten disease.
    Kwamina Nyame, Andy Hims, Aya Aburous, Nouf N Laqtom, Wentao Dong, Uche N Medoh, Julia C Heiby, Jian Xiong, Alessandro Ori, Monther Abu-Remaileh.
      Batten disease, the most prevalent form of neurodegeneration in children, is caused by mutations in the CLN3 gene, which encodes a lysosomal transmembrane protein. CLN3 loss leads to significant accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism in the lysosome. Despite GPD storage being robustly observed upon CLN3 loss, the role of GPDs in neuropathology remains unclear. Here, we demonstrate that GPDs act as potent inhibitors of glycerophospholipid catabolism in the lysosome using human cell lines and mouse models. Mechanistically, GPDs bind and competitively inhibit the lysosomal phospholipases PLA2G15 and PLBD2, which we establish to possess phospholipase B activity. GPDs effectively inhibit the rate-limiting lysophospholipase activity of these phospholipases. Consistently, lysosomes of CLN3-deficient cells and tissues accumulate toxic lysophospholipids. Our work establishes that the storage material in Batten disease directly disrupts lysosomal lipid homeostasis, suggesting GPD clearance as a potential therapeutic approach to this fatal disease.
    Keywords:  Batten disease; CLN3; GPDs; PLA2G15; PLBD2; glycerophosphodiesters; lysosomal storage disease; lysosome; neurodegeneration; phospholipase; phospholipid metabolism
    DOI:  https://doi.org/10.1016/j.molcel.2024.02.006
  10. Nat Commun. 2024 Mar 02. 15(1): 1943
    RTF2 controls replication repriming and ribonucleotide excision at the replisome.
    Brooke A Conti, Penelope D Ruiz, Cayla Broton, Nicolas J Blobel, Molly C Kottemann, Sunandini Sridhar, Francis P Lach, Tom F Wiley, Nanda K Sasi, Thomas Carroll, Agata Smogorzewska.
      DNA replication through a challenging genomic landscape is coordinated by the replisome, which must adjust to local conditions to provide appropriate replication speed and respond to lesions that hinder its progression. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2), regulate Replication Termination Factor 2 (RTF2) levels at stalled replisomes, allowing fork stabilization and restart. Here, we show that during unperturbed replication, RTF2 regulates replisome localization of RNase H2, a heterotrimeric enzyme that removes RNA from RNA-DNA heteroduplexes. RTF2, like RNase H2, is essential for mammalian development and maintains normal replication speed. However, persistent RTF2 and RNase H2 at stalled replication forks prevent efficient replication restart, which is dependent on PRIM1, the primase component of DNA polymerase α-primase. Our data show a fundamental need for RTF2-dependent regulation of replication-coupled ribonucleotide removal and reveal the existence of PRIM1-mediated direct replication restart in mammalian cells.
    DOI:  https://doi.org/10.1038/s41467-024-45947-z
  11. Mol Cell. 2024 Feb 29. pii: S1097-2765(24)00100-X. [Epub ahead of print]
    Sister chromatid cohesion halts DNA loop expansion.
    Nathalie Bastié, Christophe Chapard, Axel Cournac, Sanae Nejmi, Henri Mboumba, Olivier Gadal, Agnès Thierry, Frederic Beckouët, Romain Koszul.
      Eukaryotic genomes are folded into DNA loops mediated by structural maintenance of chromosomes (SMC) complexes such as cohesin, condensin, and Smc5/6. This organization regulates different DNA-related processes along the cell cycle, such as transcription, recombination, segregation, and DNA repair. During the G2 stage, SMC-mediated DNA loops coexist with cohesin complexes involved in sister chromatid cohesion (SCC). However, the articulation between the establishment of SCC and the formation of SMC-mediated DNA loops along the chromatin remains unknown. Here, we show that SCC is indeed a barrier to cohesin-mediated DNA loop expansion along G2/M Saccharomyces cerevisiae chromosomes.
    Keywords:  S. cerevisiae; SCC; SMC; chromatin loop; cohesin; loop extrusion; mitosis; roadblock; segregation; yeast
    DOI:  https://doi.org/10.1016/j.molcel.2024.02.004
  12. Nat Commun. 2024 Mar 06. 15(1): 2029
    Microtubule damage shapes the acetylation gradient.
    Mireia Andreu-Carbó, Cornelia Egoldt, Marie-Claire Velluz, Charlotte Aumeier.
      The properties of single microtubules within the microtubule network can be modulated through post-translational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could enter through the microtubule ends and at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen can be modulated by kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase αTAT1 is independent of kinesin-1-caused shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de)acetylation and scales with the size of the cells. The control of shaft damage represents a mechanism to regulate PTMs inside the microtubule by giving access to the lumen.
    DOI:  https://doi.org/10.1038/s41467-024-46379-5
  13. Nat Commun. 2024 Mar 07. 15(1): 2067
    A SWI/SNF-dependent transcriptional regulation mediated by POU2AF2/C11orf53 at enhancer.
    Aileen Szczepanski, Natsumi Tsuboyama, Huijue Lyu, Ping Wang, Oguzhan Beytullahoglu, Te Zhang, Benjamin David Singer, Feng Yue, Zibo Zhao, Lu Wang.
      Recent studies have identified a previously uncharacterized protein C11orf53 (now named POU2AF2/OCA-T1), which functions as a robust co-activator of POU2F3, the master transcription factor which is critical for both normal and neoplastic tuft cell identity and viability. Here, we demonstrate that POU2AF2 dictates opposing transcriptional regulation at distal enhance elements. Loss of POU2AF2 leads to an inhibition of active enhancer nearby genes, such as tuft cell identity genes, and a derepression of Polycomb-dependent poised enhancer nearby genes, which are critical for cell viability and differentiation. Mechanistically, depletion of POU2AF2 results in a global redistribution of the chromatin occupancy of the SWI/SNF complex, leading to a significant 3D genome structure change and a subsequent transcriptional reprogramming. Our genome-wide CRISPR screen further demonstrates that POU2AF2 depletion or SWI/SNF inhibition leads to a PTEN-dependent cell growth defect, highlighting a potential role of POU2AF2-SWI/SNF axis in small cell lung cancer (SCLC) pathogenesis. Additionally, pharmacological inhibition of SWI/SNF phenocopies POU2AF2 depletion in terms of gene expression alteration and cell viability decrease in SCLC-P subtype cells. Therefore, impeding POU2AF2-mediated transcriptional regulation represents a potential therapeutic approach for human SCLC therapy.
    DOI:  https://doi.org/10.1038/s41467-024-46492-5
  14. J Cell Biol. 2024 Apr 01. pii: e202306142. [Epub ahead of print]223(4):
    Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength.
    Manolo U Rios, Małgorzata A Bagnucka, Bryan D Ryder, Beatriz Ferreira Gomes, Nicole E Familiari, Kan Yaguchi, Matthew Amato, Weronika E Stachera, Łukasz A Joachimiak, Jeffrey B Woodruff.
      The outermost layer of centrosomes, called pericentriolar material (PCM), organizes microtubules for mitotic spindle assembly. The molecular interactions that enable PCM to assemble and resist external forces are poorly understood. Here, we use crosslinking mass spectrometry (XL-MS) to analyze PLK-1-potentiated multimerization of SPD-5, the main PCM scaffold protein in C. elegans. In the unassembled state, SPD-5 exhibits numerous intramolecular crosslinks that are eliminated after phosphorylation by PLK-1. Thus, phosphorylation induces a structural opening of SPD-5 that primes it for assembly. Multimerization of SPD-5 is driven by interactions between multiple dispersed coiled-coil domains. Structural analyses of a phosphorylated region (PReM) in SPD-5 revealed a helical hairpin that dimerizes to form a tetrameric coiled-coil. Mutations within this structure and other interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces, revealing that PCM assembly and strength are interdependent. We propose that PCM size and strength emerge from specific, multivalent coiled-coil interactions between SPD-5 proteins.
    DOI:  https://doi.org/10.1083/jcb.202306142
  15. J Cell Biol. 2024 May 06. pii: e202303141. [Epub ahead of print]223(5):
    DUX4-induced HSATII transcription causes KDM2A/B-PRC1 nuclear foci and impairs DNA damage response.
    Tessa Arends, Hiroshi Tsuchida, Richard O Adeyemi, Stephen J Tapscott.
      Polycomb repressive complexes regulate developmental gene programs, promote DNA damage repair, and mediate pericentromeric satellite repeat repression. Expression of pericentromeric satellite repeats has been implicated in several cancers and diseases, including facioscapulohumeral dystrophy (FSHD). Here, we show that DUX4-mediated transcription of HSATII regions causes nuclear foci formation of KDM2A/B-PRC1 complexes, resulting in a global loss of PRC1-mediated monoubiquitination of histone H2A. Loss of PRC1-ubiquitin signaling severely impacts DNA damage response. Our data implicate DUX4-activation of HSATII and sequestration of KDM2A/B-PRC1 complexes as a mechanism of regulating epigenetic and DNA repair pathways.
    DOI:  https://doi.org/10.1083/jcb.202303141
  16. Nat Struct Mol Biol. 2024 Mar 08.
    Structure of the human KMN complex and implications for regulation of its assembly.
    Soumitra Polley, Tobias Raisch, Sabrina Ghetti, Marie Körner, Melina Terbeck, Frauke Gräter, Stefan Raunser, Camilo Aponte-Santamaría, Ingrid R Vetter, Andrea Musacchio.
      Biorientation of chromosomes during cell division is necessary for precise dispatching of a mother cell's chromosomes into its two daughters. Kinetochores, large layered structures built on specialized chromosome loci named centromeres, promote biorientation by binding and sensing spindle microtubules. One of the outer layer main components is a ten-subunit assembly comprising Knl1C, Mis12C and Ndc80C (KMN) subcomplexes. The KMN is highly elongated and docks on kinetochores and microtubules through interfaces at its opposite extremes. Here, we combine cryogenic electron microscopy reconstructions and AlphaFold2 predictions to generate a model of the human KMN that reveals all intra-KMN interfaces. We identify and functionally validate two interaction interfaces that link Mis12C to Ndc80C and Knl1C. Through targeted interference experiments, we demonstrate that this mutual organization strongly stabilizes the KMN assembly. Our work thus reports a comprehensive structural and functional analysis of this part of the kinetochore microtubule-binding machinery and elucidates the path of connections from the chromatin-bound components to the force-generating components.
    DOI:  https://doi.org/10.1038/s41594-024-01230-9
  17. Cell Rep. 2024 Mar 06. pii: S2211-1247(24)00191-8. [Epub ahead of print]43(3): 113863
    Integrated stress response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation.
    Lydia Grmai, Manuel Michaca, Emily Lackner, Narayanan Nampoothiri V P, Deepika Vasudevan.
      Reproduction is an energy-intensive process requiring systemic coordination. However, the inter-organ signaling mechanisms that relay nutrient status to modulate reproductive output are poorly understood. Here, we use Drosophila melanogaster as a model to establish the integrated stress response (ISR) transcription factor, Atf4, as a fat tissue metabolic sensor that instructs oogenesis. We demonstrate that Atf4 regulates lipase activity to mediate yolk lipoprotein synthesis in the fat body. Depletion of Atf4 in the fat body also blunts oogenesis recovery after amino acid deprivation and re-feeding, suggestive of a nutrient-sensing role for Atf4. We also discovered that Atf4 promotes secretion of a fat-body-derived neuropeptide, CNMamide, which modulates neural circuits that promote egg-laying behavior (ovulation). Thus, we posit that ISR signaling in fat tissue acts as a "metabolic sensor" that instructs female reproduction-directly by impacting yolk lipoprotein production and follicle maturation and systemically by regulating ovulation.
    Keywords:  Atf4; CNMa; CP: Cell biology; CP: Developmental biology; adipocyte; bmm; egg retention; integrated stress response; oogenesis; ovulation; yolk protein
    DOI:  https://doi.org/10.1016/j.celrep.2024.113863
  18. Nat Aging. 2024 Mar 04.
    Tissue mosaicism following stem cell aging: blood as an exemplar.
    Chiraag D Kapadia, Margaret A Goodell.
      Loss of stem cell regenerative potential underlies aging of all tissues. Somatic mosaicism, the emergence of cellular patchworks within tissues, increases with age and has been observed in every organ yet examined. In the hematopoietic system, as in most tissues, stem cell aging through a variety of mechanisms occurs in lockstep with the emergence of somatic mosaicism. Here, we draw on insights from aging hematopoiesis to illustrate fundamental principles of stem cell aging and somatic mosaicism. We describe the generalizable changes intrinsic to aged stem cells and their milieu that provide the backdrop for somatic mosaicism to emerge. We discuss genetic and nongenetic mechanisms that can result in tissue somatic mosaicism and existing methodologies to detect such clonal outgrowths. Finally, we propose potential avenues to modify mosaicism during aging, with the ultimate aim of increasing tissue resiliency.
    DOI:  https://doi.org/10.1038/s43587-024-00589-0
  19. Cell. 2024 Mar 01. pii: S0092-8674(24)00180-6. [Epub ahead of print]
    Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle.
    Athanasios Litsios, Benjamin T Grys, Oren Z Kraus, Helena Friesen, Catherine Ross, Myra Paz David Masinas, Duncan T Forster, Mary T Couvillion, Stefanie Timmermann, Maximilian Billmann, Chad Myers, Nils Johnsson, L Stirling Churchman, Charles Boone, Brenda J Andrews.
      Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of ∼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.
    Keywords:  Saccharomyces cerevisiae; automated image analysis; cell cycle; deep learning; differential scaling; high-content screening; protein localization; proteomics; spatiotemporal proteome; systems biology
    DOI:  https://doi.org/10.1016/j.cell.2024.02.014
  20. Nat Commun. 2024 Mar 05. 15(1): 1992
    In-cell NMR suggests that DNA i-motif levels are strongly depleted in living human cells.
    Pavlína Víšková, Eva Ištvánková, Jan Ryneš, Šimon Džatko, Tomáš Loja, Martina Lenarčič Živković, Riccardo Rigo, Roberto El-Khoury, Israel Serrano-Chacón, Masad J Damha, Carlos González, Jean-Louis Mergny, Silvie Foldynová-Trantírková, Lukáš Trantírek.
      I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitor iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37 °C. Our findings show that iMFPS displaying pHT < 7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT > 7 appear as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) reveals that cell cycle-dependent iM formation has a dual origin, and iM formation concerns only a tiny fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.
    DOI:  https://doi.org/10.1038/s41467-024-46221-y
  21. Cell Stem Cell. 2024 Mar 07. pii: S1934-5909(24)00047-X. [Epub ahead of print]31(3): 359-377.e10
    A mitochondrial NADPH-cholesterol axis regulates extracellular vesicle biogenesis to support hematopoietic stem cell fate.
    Massimo Bonora, Claudia Morganti, Nick van Gastel, Kyoko Ito, Enrica Calura, Ilaria Zanolla, Letizia Ferroni, Yang Zhang, Yookyung Jung, Gabriele Sales, Paolo Martini, Takahisa Nakamura, Francesco Massimo Lasorsa, Toren Finkel, Charles P Lin, Barbara Zavan, Paolo Pinton, Irene Georgakoudi, Chiara Romualdi, David T Scadden, Keisuke Ito.
      Mitochondrial fatty acid oxidation (FAO) is essential for hematopoietic stem cell (HSC) self-renewal; however, the mechanism by which mitochondrial metabolism controls HSC fate remains unknown. Here, we show that within the hematopoietic lineage, HSCs have the largest mitochondrial NADPH pools, which are required for proper HSC cell fate and homeostasis. Bioinformatic analysis of the HSC transcriptome, biochemical assays, and genetic inactivation of FAO all indicate that FAO-generated NADPH fuels cholesterol synthesis in HSCs. Interference with FAO disturbs the segregation of mitochondrial NADPH toward corresponding daughter cells upon single HSC division. Importantly, we have found that the FAO-NADPH-cholesterol axis drives extracellular vesicle (EV) biogenesis and release in HSCs, while inhibition of EV signaling impairs HSC self-renewal. These data reveal the existence of a mitochondrial NADPH-cholesterol axis for EV biogenesis that is required for hematopoietic homeostasis and highlight the non-stochastic nature of HSC fate determination.
    Keywords:  HSC self-renewal; NADPH; cholesterol; exosomes; extracellular vesicles; fate determination; fatty acid oxidation; hematopoietic stem cell; metabolism; mitochondria
    DOI:  https://doi.org/10.1016/j.stem.2024.02.004
  22. J Clin Invest. 2024 Mar 07. pii: e173371. [Epub ahead of print]
    An in vivo screening platform identifies senolytic compounds that target p16INK4a+ fibroblasts in lung fibrosis.
    Jin Young Lee, Nabora S Reyes, Supriya Ravishankar, Minqi Zhou, Maria Krasilnikov, Christian Ringler, Grace Pohan, Chris Wilson, Kenny Kean-Hooi Ang, Paul J Wolters, Tatsuya Tsukui, Dean Sheppard, Michelle R Arkin, Tien Peng.
      The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues ("senolytics"). However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of p16Ink4a+ cells in diseased tissues. By high-throughput screening in vitro, precision cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor (XL888) as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic p16Ink4a+ fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted p16INK4a-high human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced p16INK4a+ fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where p16INK4a+ cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mouse and human respectively and provides a senolytic screening platform for other age-related diseases.
    Keywords:  Aging; Cellular senescence; Drug screens; Fibrosis; Pulmonology
    DOI:  https://doi.org/10.1172/JCI173371
  23. Genes Dev. 2024 Mar 07.
    A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes, as well as failure of epigenetic reprogramming and zygotic genome activation in embryos.
    Carlo Giaccari, Francesco Cecere, Lucia Argenziano, Angela Pagano, Antonio Galvao, Dario Acampora, Gianna Rossi, Bruno Hay Mele, Basilia Acurzio, Scott Coonrod, Maria Vittoria Cubellis, Flavia Cerrato, Simon Andrews, Sandra Cecconi, Gavin Kelsey, Andrea Riccio.
      Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a Padi6 missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of Padi6 mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from Padi6 mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.
    Keywords:  DNMT1 localization; Padi6; epigenetic reprogramming; imprinting disorders; maternal-effect genes; multilocus imprinting disturbance; subcortical maternal complex; zygotic genome activation
    DOI:  https://doi.org/10.1101/gad.351238.123
  24. Nat Cell Biol. 2024 Mar 05.
    Distinct pathways drive anterior hypoblast specification in the implanting human embryo.
    Bailey A T Weatherbee, Antonia Weberling, Carlos W Gantner, Lisa K Iwamoto-Stohl, Zoe Barnikel, Amy Barrie, Alison Campbell, Paula Cunningham, Cath Drezet, Panagiota Efstathiou, Simon Fishel, Sandra Gutiérrez Vindel, Megan Lockwood, Rebecca Oakley, Catherine Pretty, Nabiha Chowdhury, Lucy Richardson, Anastasia Mania, Lauren Weavers, Leila Christie, Kay Elder, Phillip Snell, Magdalena Zernicka-Goetz.
      Development requires coordinated interactions between the epiblast, which generates the embryo proper; the trophectoderm, which generates the placenta; and the hypoblast, which forms both the anterior signalling centre and the yolk sac. These interactions remain poorly understood in human embryogenesis because mechanistic studies have only recently become possible. Here we examine signalling interactions post-implantation using human embryos and stem cell models of the epiblast and hypoblast. We find anterior hypoblast specification is NODAL dependent, as in the mouse. However, while BMP inhibits anterior signalling centre specification in the mouse, it is essential for its maintenance in human. We also find contrasting requirements for BMP in the naive pre-implantation epiblast of mouse and human embryos. Finally, we show that NOTCH signalling is important for human epiblast survival. Our findings of conserved and species-specific factors that drive these early stages of embryonic development highlight the strengths of comparative species studies.
    DOI:  https://doi.org/10.1038/s41556-024-01367-1
  25. Nature. 2024 Mar 06.
    Synthetic reversed sequences reveal default genomic states.
    Brendan R Camellato, Ran Brosh, Hannah J Ashe, Matthew T Maurano, Jef D Boeke.
      Pervasive transcriptional activity is observed across diverse species. The genomes of extant organisms have undergone billions of years of evolution, making it unclear whether these genomic activities represent effects of selection or 'noise'1-4. Characterizing default genome states could help understand whether pervasive transcriptional activity has biological meaning. Here we addressed this question by introducing a synthetic 101-kb locus into the genomes of Saccharomyces cerevisiae and Mus musculus and characterizing genomic activity. The locus was designed by reversing but not complementing human HPRT1, including its flanking regions, thus retaining basic features of the natural sequence but ablating evolved coding or regulatory information. We observed widespread activity of both reversed and native HPRT1 loci in yeast, despite the lack of evolved yeast promoters. By contrast, the reversed locus displayed no activity at all in mouse embryonic stem cells, and instead exhibited repressive chromatin signatures. The repressive signature was alleviated in a locus variant lacking CpG dinucleotides; nevertheless, this variant was also transcriptionally inactive. These results show that synthetic genomic sequences that lack coding information are active in yeast, but inactive in mouse embryonic stem cells, consistent with a major difference in 'default genomic states' between these two divergent eukaryotic cell types, with implications for understanding pervasive transcription, horizontal transfer of genetic information and the birth of new genes.
    DOI:  https://doi.org/10.1038/s41586-024-07128-2
  26. Nat Commun. 2024 Mar 08. 15(1): 2132
    GRB2 stabilizes RAD51 at reversed replication forks suppressing genomic instability and innate immunity against cancer.
    Zu Ye, Shengfeng Xu, Yin Shi, Xueqian Cheng, Yuan Zhang, Sunetra Roy, Sarita Namjoshi, Michael A Longo, Todd M Link, Katharina Schlacher, Guang Peng, Dihua Yu, Bin Wang, John A Tainer, Zamal Ahmed.
      Growth factor receptor-bound protein 2 (GRB2) is a cytoplasmic adapter for tyrosine kinase signaling and a nuclear adapter for homology-directed-DNA repair. Here we find nuclear GRB2 protects DNA at stalled replication forks from MRE11-mediated degradation in the BRCA2 replication fork protection axis. Mechanistically, GRB2 binds and inhibits RAD51 ATPase activity to stabilize RAD51 on stalled replication forks. In GRB2-depleted cells, PARP inhibitor (PARPi) treatment releases DNA fragments from stalled forks into the cytoplasm that activate the cGAS-STING pathway to trigger pro-inflammatory cytokine production. Moreover in a syngeneic mouse metastatic ovarian cancer model, GRB2 depletion in the context of PARPi treatment reduced tumor burden and enabled high survival consistent with immune suppression of cancer growth. Collective findings unveil GRB2 function and mechanism for fork protection in the BRCA2-RAD51-MRE11 axis and suggest GRB2 as a potential therapeutic target and an enabling predictive biomarker for patient selection for PARPi and immunotherapy combination.
    DOI:  https://doi.org/10.1038/s41467-024-46283-y
  27. Genome Biol. 2024 Mar 04. 25(1): 62
    scAbsolute: measuring single-cell ploidy and replication status.
    Michael P Schneider, Amy E Cullen, Justina Pangonyte, Jason Skelton, Harvey Major, Elke Van Oudenhove, Maria J Garcia, Blas Chaves Urbano, Anna M Piskorz, James D Brenton, Geoff Macintyre, Florian Markowetz.
      Cancer cells often exhibit DNA copy number aberrations and can vary widely in their ploidy. Correct estimation of the ploidy of single-cell genomes is paramount for downstream analysis. Based only on single-cell DNA sequencing information, scAbsolute achieves accurate and unbiased measurement of single-cell ploidy and replication status, including whole-genome duplications. We demonstrate scAbsolute's capabilities using experimental cell multiplets, a FUCCI cell cycle expression system, and a benchmark against state-of-the-art methods. scAbsolute provides a robust foundation for single-cell DNA sequencing analysis across different technologies and has the potential to enable improvements in a number of downstream analyses.
    Keywords:  Cell cycle stage; Ploidy estimation; Singe-cell genomics; Single-cell DNA sequencing; Whole-genome doubling; Whole-genome duplication
    DOI:  https://doi.org/10.1186/s13059-024-03204-y
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