bims-ginsta Biomed News
on Genome instability
Issue of 2023‒11‒26
27 papers selected by
Jinrong Hu, National University of Singapore

  1. Mol Cell. 2023 Nov 14. pii: S1097-2765(23)00868-7. [Epub ahead of print]
      DEAD-box ATPases are major regulators of biomolecular condensates and orchestrate diverse biochemical processes that are critical for the functioning of cells. How DEAD-box proteins are selectively recruited to their respective biomolecular condensates is unknown. We explored this in the context of the nucleolus and DEAD-box protein DDX21. We find that the pH of the nucleolus is intricately linked to the transcriptional activity of the organelle and facilitates the recruitment and condensation of DDX21. We identify an evolutionarily conserved feature of the C terminus of DDX21 responsible for nucleolar localization. This domain is essential for zebrafish development, and its intrinsically disordered and isoelectric properties are necessary and sufficient for the ability of DDX21 to respond to changes in pH and form condensates. Molecularly, the enzymatic activities of poly(ADP-ribose) polymerases contribute to maintaining the nucleolar pH and, consequently, DDX21 recruitment and nucleolar partitioning. These observations reveal an activity-dependent physicochemical mechanism for the selective recruitment of biochemical activities to biomolecular condensates.
    Keywords:  DEAD-box proteins; PARP; RNA; biomolecular condensates; intrinsically disordered regions; nucleolus; pH; zebrafish development
  2. bioRxiv. 2023 Nov 10. pii: 2023.11.08.566322. [Epub ahead of print]
      Terminal differentiation requires a massive restructuring of the transcriptome. During intestinal differentiation, the expression patterns of nearly 4000 genes are altered as cells transition from progenitor cells in crypts to differentiated cells in villi. We identified dynamic recruitment of RNA Polymerase II (Pol II) to gene promoters as the primary driver of transcriptomic shifts during intestinal differentiation in vivo . Changes in enhancer-promoter looping interactions accompany dynamic Pol II recruitment and are dependent upon HNF4, a pro-differentiation transcription factor. Using genetic loss-of-function, ChIP-seq and IP mass spectrometry, we demonstrate that HNF4 collaborates with chromatin remodelers and loop-stabilizing proteins and facilitates Pol II recruitment at hundreds of genes pivotal to differentiation. We also explore alternate mechanisms which drive differentiation gene expression and find pause-release of Pol II and post-transcriptional mRNA stability regulate smaller subsets of differentially expressed genes. These studies provide insights into the mechanisms of differentiation in a renewing adult tissue.HIGHLIGHTS: Dynamic recruitment of Pol II largely drives the vast transcriptomic changes seen during differentiation of mouse intestinal epithelium.Smaller groups of differentiated genes are subject to regulation through Pol II pause-release and post-transcriptional mechanisms such as differences in mRNA stability.IP-mass spectrometry analysis identifies the first interactome of HNF4 in the differentiated small intestine, finding interactions with chromatin looping and chromatin remodeling proteins.HNF4 transcription factors play a critical role in recruiting Pol II to the promoters of essential intestinal differentiation genes.
  3. Cell. 2023 Nov 22. pii: S0092-8674(23)01174-1. [Epub ahead of print]186(24): 5269-5289.e22
      A generic level of chromatin organization generated by the interplay between cohesin and CTCF suffices to limit promiscuous interactions between regulatory elements, but a lineage-specific chromatin assembly that supersedes these constraints is required to configure the genome to guide gene expression changes that drive faithful lineage progression. Loss-of-function approaches in B cell precursors show that IKAROS assembles interactions across megabase distances in preparation for lymphoid development. Interactions emanating from IKAROS-bound enhancers override CTCF-imposed boundaries to assemble lineage-specific regulatory units built on a backbone of smaller invariant topological domains. Gain of function in epithelial cells confirms IKAROS' ability to reconfigure chromatin architecture at multiple scales. Although the compaction of the Igκ locus required for genome editing represents a function of IKAROS unique to lymphocytes, the more general function to preconfigure the genome to support lineage-specific gene expression and suppress activation of extra-lineage genes provides a paradigm for lineage restriction.
    Keywords:  3D genome organization; Hi-C; HiChIP; IKAROS; Igκ locus contraction; enhancer loops; inter-compartmental loops; lineage-specific genome organization; lymphocyte development; superTADs
  4. Cell Rep. 2023 Nov 22. pii: S2211-1247(23)01507-3. [Epub ahead of print]42(12): 113495
      Nuclear envelope (NE) disassembly during mitosis is critical to ensure faithful segregation of the genetic material. NE disassembly is a phosphorylation-dependent process wherein mitotic kinases hyper-phosphorylate lamina and nucleoporins to initiate nuclear envelope breakdown (NEBD). In this study, we uncover an unexpected role of the PP2A phosphatase B55SUR-6 in NEBD during the first embryonic division of Caenorhabditis elegans embryo. B55SUR-6 depletion delays NE permeabilization and stabilizes lamina and nucleoporins. As a result, the merging of parental genomes and chromosome segregation is impaired. NEBD defect upon B55SUR-6 depletion is not due to delayed mitotic onset or mislocalization of mitotic kinases. Importantly, we demonstrate that microtubule-dependent mechanical forces synergize with B55SUR-6 for efficient NEBD. Finally, our data suggest that the lamin LMN-1 is likely a bona fide target of PP2A-B55SUR-6. These findings establish a model highlighting biochemical crosstalk between kinases, PP2A-B55SUR-6 phosphatase, and microtubule-generated mechanical forces in timely NE dissolution.
    Keywords:  Aurora A; C. elegans; CDK-1; CP: Cell biology; PLK-1; PP2A-B55; dynein; lamina; microtubules; nuclear envelope breakdown (NEBD); nucleoporins
  5. Cell Rep. 2023 Nov 22. pii: S2211-1247(23)01469-9. [Epub ahead of print]42(12): 113457
      While programmed cell death plays important roles during morphogenetic stages of development, post-differentiation organ growth is considered an efficient process whereby cell proliferation increases cell number. Here we demonstrate that early postnatal growth of the pancreas unexpectedly involves massive acinar cell elimination. Measurements of cell proliferation and death in the human pancreas in comparison to the actual increase in cell number predict daily elimination of 0.7% of cells, offsetting 88% of cell formation over the first year of life. Using mouse models, we show that death is associated with mitosis, through a failure of dividing cells to generate two viable daughters. In p53-deficient mice, acinar cell death and proliferation are reduced, while organ size is normal, suggesting that p53-dependent developmental apoptosis triggers compensatory proliferation. We propose that excess cell turnover during growth of the pancreas, and presumably other organs, facilitates robustness to perturbations and supports maintenance of tissue architecture.
    Keywords:  CP: Developmental biology; acinar cells; compensatory proliferation; p53; pancreas; postnatal development; programmed cell death
  6. Stem Cell Reports. 2023 Nov 08. pii: S2213-6711(23)00420-4. [Epub ahead of print]
      Histone 3 lysine 79 methylation (H3K79me) is enriched on gene bodies proportional to gene expression levels and serves as a strong barrier for the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). DOT1L is the sole histone methyltransferase that deposits all three orders-mono (me1), di (me2), and tri (me3) methylation-at H3K79. Here, we leverage genetic and chemical approaches to parse the specific functions of orders of H3K79me in maintaining cell identity. DOT1L interacts with AF10 (Mllt10), which recognizes unmodified H3K27 and boosts H3K79me2/3 methylation. AF10 deletion evicts H3K79me2/3 and reorganizes H3K79me1 to the transcription start site to facilitate iPSC formation in the absence of steady-state transcriptional changes. Instead, AF10 loss redistributes RNA polymerase II to a uniquely pluripotent pattern at highly expressed, rapidly transcribed housekeeping genes. Taken together, we reveal a specific mechanism for H3K79me2/3 located at the gene body in reinforcing cell identity.
  7. bioRxiv. 2023 Nov 11. pii: 2023.11.11.566679. [Epub ahead of print]
      Genomic information must be faithfully transmitted into two daughter cells during cell division. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase IIα are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Macromolecular crowding/depletion force, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the "chromosome milieu" during mitosis of living human cells using orientation-independent-differential interference contrast (OI-DIC) module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prometaphase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. In vitro , native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like, with further condensation. These results suggest that a transient rise in macromolecular crowding (proteins and RNAs), likely triggered by the relocation of macromolecules via nuclear envelope breakdown and also by a subsequent decrease in cell-volumes, contributes to mitotic chromosome condensation, shedding light on a new aspect of the condensation mechanism in living human cells.Significance Statement: Mitotic chromosome condensation is an essential process to transmit replicated chromosomes into two daughter cells during cell division. To study the underlying physical principles of this process, we focused on macromolecular crowding/depletion force, which is a force that attracts large structures in crowded cell environments. Using newly developed special light microscopy, which can image the molecular density of cellular environments, we found that crowding around chromosomes increases during cell division. In vitro , higher concentrations of macromolecules condense chromatin and make it stiffer and more solid-like. Our results suggest that the rise in macromolecular crowding renders chromosomes more rigid, ensuring accurate chromosome transmission during cell division.
  8. Fertil Steril. 2023 Nov 20. pii: S0015-0282(23)02001-0. [Epub ahead of print]
      The oocyte, a long-lived, post-mitotic cell, is the locus of reproductive aging in women. Female germ cells replicate only during fetal life and age throughout reproductive life. Mechanisms of oocyte aging include accumulation of oxidative damage, mitochondrial dysfunction and disruption of proteins, including cohesion. Nobel Laureate Bob Edwards also discovered a "production line" during oogonial replication in the mouse, wherein the last oocytes to ovulate in the adult derived from the last oogonia to exit mitotic replication in the fetus. Based on this, we proposed a two hit "telomere theory of reproductive aging" to integrate the myriad features of oocyte aging. The first hit- oocytes remaining in older women traversed more cell cycles during fetal oogenesis. The second hit- oocytes accumulated more environmental and endogenous oxidative damage across the life of the woman. Telomeres could mediate both these aspects of oocyte aging. Telomeres provide a "mitotic clock", with telomere attrition an inevitable consequence of cell division due to of the end-replication problem. And telomere's guanine-rich sequence renders them especially sensitive to oxidative damage, even in post-mitotic cells. Telomerase, the reverse transcriptase that restores telomeres, is better at maintaining than elongating telomeres. Moreover, telomerase remains inactive during much of oogenesis and early development. Oocytes are left with short telomeres, on the brink of viability. In support of this theory, mice with induced telomere attrition and women with naturally occurring telomeropathy suffer diminished ovarian reserve, abnormal embryo development and infertility. In contrast, sperm are produced throughout the life of the male by a telomerase-active progenitor, spermatogonia, resulting in the longest telomeres in the body. In mice, cleavage stage embryos elongate telomeres via "Alternative Lengthening of Telomeres (ALT)", a recombination-based mechanism, rarely encountered outside of telomerase-deficient cancers. Many questions about telomeres and reproduction are raised by these findings:- Does the "normal" telomere attrition observed in human oocytes contribute to their extraordinarily high rate of meiotic non-disjunction? Does recombination-based telomere elongation render embryos susceptible to mitotic non-disjunction (and mosaicism)? Can some features of telomeres serve as markers of oocyte quality?
    Keywords:  Telomeres; embryos; infertility; oocytes; telomerase
  9. bioRxiv. 2023 Nov 06. pii: 2023.11.06.565883. [Epub ahead of print]
      Actin cortex patterning and dynamics are critical for cell shape changes. These dynamics undergo transitions during development, often accompanying changes in collective cell behavior. While mechanisms have been established for individual cells' dynamic behaviors, mechanisms and specific molecules that result in developmental transitions in vivo are still poorly understood. Here, we took advantage of two developmental systems in Drosophila melanogaster to identify conditions that altered cortical patterning and dynamics. We identified a RhoGEF and RhoGAP pair whose relocalization from nucleus to cortex results in actomyosin waves in egg chambers. Furthermore, we found that overexpression of a different RhoGEF and RhoGAP pair resulted in actomyosin waves in the early embryo, during which RhoA activation precedes actomyosin assembly and RhoGAP recruitment by ∼4 seconds. Overall, we showed a mechanism involved in inducing actomyosin waves that is essential for oocyte development and is general to other cell types.
  10. bioRxiv. 2023 Nov 11. pii: 2023.11.11.566681. [Epub ahead of print]
      Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the roles of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks to many neurologically relevant mRNAs in NPCs. Our data reveal eIF3 predominantly interacts with 3' untranslated region (3'-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. High eIF3 crosslinking at 3'-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling. We identify the transcriptional regulator inhibitor of DNA binding 2 ( ID2 ) mRNA as a case in which active translation levels and eIF3 crosslinking are dramatically increased upon early NPC differentiation. Furthermore, we find that eIF3 engagement at 3'-UTR ends is dependent on polyadenylation. The results presented here show that eIF3 engages with 3'-UTR termini of highly translated mRNAs, supporting a role of mRNA circularization in the mechanisms governing mRNA translation in NPCs.
  11. bioRxiv. 2023 Nov 11. pii: 2023.11.07.566063. [Epub ahead of print]
      Nuclear atypia, including altered nuclear size, contour, and chromatin organization, is ubiquitous in cancer cells. Atypical primary nuclei and micronuclei can rupture during interphase; however, the frequency, causes, and consequences of nuclear rupture are unknown in most cancers. We demonstrate that nuclear envelope rupture is surprisingly common in many human cancers, particularly glioblastoma. Using highly-multiplexed 2D and super-resolution 3D-imaging of glioblastoma tissues and patient-derived xenografts and cells, we link primary nuclear rupture with reduced lamin A/C and micronuclear rupture with reduced lamin B1. Moreover, ruptured glioblastoma cells activate cGAS-STING-signaling involved in innate immunity. We observe that local patterning of cell states influences tumor spatial organization and is linked to both lamin expression and rupture frequency, with neural-progenitor-cell-like states exhibiting the lowest lamin A/C levels and greatest susceptibility to primary nuclear rupture. Our study reveals that nuclear instability is a core feature of cancer, and links nuclear integrity, cell state, and immune signaling.
  12. Nat Commun. 2023 Nov 20. 14(1): 7564
      Even slight imbalance between the growth rate of different organs can accumulate to a large deviation from their appropriate size during development. Here, we use live imaging of the pharynx of C. elegans to ask if and how organ size scaling nevertheless remains uniform among individuals. Growth trajectories of hundreds of individuals reveal that pharynxes grow by a near constant volume per larval stage that is independent of their initial size, such that undersized pharynxes catch-up in size during development. Tissue-specific depletion of RAGA-1, an activator of mTOR and growth, shows that maintaining correct pharynx-to-body size proportions involves a bi-directional coupling between pharynx size and body growth. In simulations, this coupling cannot be explained by limitation of food uptake alone, and genetic experiments reveal an involvement of the mechanotransducing transcriptional co-regulator yap-1. Our data suggests that mechanotransduction coordinates pharynx growth with other tissues, ensuring body plan uniformity among individuals.
  13. EMBO J. 2023 Nov 21. e114221
      Efficient treatment of acute myeloid leukemia (AML) patients remains a challenge despite recent therapeutic advances. Here, using a CRISPRi screen targeting chromatin factors, we identified the nucleosome-remodeling factor (NURF) subunit BPTF as an essential regulator of AML cell survival. We demonstrate that BPTF forms an alternative NURF chromatin remodeling complex with SMARCA5 and BAP18, which regulates the accessibility of a large set of insulator regions in leukemic cells. This ensures efficient CTCF binding and boundary formation between topologically associated domains that is essential for maintaining the leukemic transcriptional programs. We also demonstrate that the well-studied PHD2-BROMO chromatin reader domains of BPTF, while contributing to complex recruitment to chromatin, are dispensable for leukemic cell growth. Taken together, our results uncover how the alternative NURF complex contributes to leukemia and provide a rationale for its targeting in AML.
    Keywords:  BPTF; SMARCA5; acute myeloid leukemia; chromatin remodeling; insulator regions
  14. Trends Endocrinol Metab. 2023 Nov 21. pii: S1043-2760(23)00237-0. [Epub ahead of print]
      Cardiac macrophages are essential mediators of cardiac development, tissue homeostasis, and response to injury. Cell-intrinsic shifts in metabolism and availability of metabolites regulate macrophage function. The human and mouse heart contain a heterogeneous compilation of cardiac macrophages that are derived from at least two distinct lineages. In this review, we detail the unique functional roles and metabolic profiles of tissue-resident and monocyte-derived cardiac macrophages during embryonic development and adult tissue homeostasis and in response to pathologic and physiologic stressors. We discuss the metabolic preferences of each macrophage lineage and how metabolism influences monocyte fate specification. Finally, we highlight the contribution of cardiac macrophages and derived metabolites on cell-cell communication, metabolic health, and disease pathogenesis.
    Keywords:  HFpEF; cardiac; exercise; infarction; macrophage; metabolism
  15. Nucleic Acids Res. 2023 Nov 24. pii: gkad1122. [Epub ahead of print]
      RNA-binding proteins (RBPs) with intrinsically disordered regions (IDRs) are linked to multiple human disorders, but their mechanisms of action remain unclear. Here, we report that one such protein, Nocte, is essential for Drosophila eye development by regulating a critical gene expression cascade at translational level. Knockout of nocte in flies leads to lethality, and its eye-specific depletion impairs eye size and morphology. Nocte preferentially enhances translation of mRNAs with long upstream open reading frames (uORFs). One of the key Nocte targets, glass mRNA, encodes a transcription factor critical for differentiation of photoreceptor neurons and accessory cells, and re-expression of Glass largely rescued the eye defects caused by Nocte depletion. Mechanistically, Nocte counteracts long uORF-mediated translational suppression by promoting translation reinitiation downstream of the uORF. Nocte interacts with translation factors eIF3 and Rack1 through its BAT2 domain, and a Nocte mutant lacking this domain fails to promote translation of glass mRNA. Notably, de novo mutations of human orthologs of Nocte have been detected in schizophrenia patients. Our data suggest that Nocte family of proteins can promote translation reinitiation to overcome long uORFs-mediated translational suppression, and disruption of this function can lead to developmental defects and neurological disorders.
  16. EMBO Rep. 2023 Nov 20. e57910
      Protein translocation across the endoplasmic reticulum (ER) membrane is an essential step during protein entry into the secretory pathway. The conserved Sec61 protein-conducting channel facilitates polypeptide translocation and coordinates cotranslational polypeptide-processing events. In cells, the majority of Sec61 is stably associated with a heterotetrameric membrane protein complex, the translocon-associated protein complex (TRAP), yet the mechanism by which TRAP assists in polypeptide translocation remains unknown. Here, we present the structure of the core Sec61/TRAP complex bound to a mammalian ribosome by cryogenic electron microscopy (cryo-EM). Ribosome interactions anchor the Sec61/TRAP complex in a conformation that renders the ER membrane locally thinner by significantly curving its lumenal leaflet. We propose that TRAP stabilizes the ribosome exit tunnel to assist nascent polypeptide insertion through Sec61 and provides a ratcheting mechanism into the ER lumen mediated by direct polypeptide interactions.
    Keywords:  cryo-EM; membrane proteins; protein translocation; secretory proteins; structural biology
  17. bioRxiv. 2023 Nov 06. pii: 2023.11.06.565717. [Epub ahead of print]
      The rete ovarii (RO) is an epithelial structure that arises during fetal development in close proximity to the ovary and persists throughout adulthood in mice. However, the functional significance of the RO remains elusive, and it has been absent from recent discussions of female reproductive anatomy. The RO comprises three distinct regions: the intraovarian rete (IOR) within the ovary, the extraovarian rete (EOR) in the periovarian tissue, and the connecting rete (CR) linking the EOR and IOR. We hypothesize that the RO plays a pivotal role in maintaining ovarian homeostasis and responding to physiological changes. To uncover the nature and function of RO cells, we conducted transcriptome analysis, encompassing bulk, single-cell, and nucleus-level sequencing of both fetal and adult RO tissues using the Pax8-rtTA; Tre- H2B-GFP mouse line, where all RO regions express nuclear GFP. This study presents three datasets, which highlight RO-specific gene expression signatures and reveal differences in gene expression across the three RO regions during development and in adulthood. The integration and rigorous validation of these datasets will advance our understanding of the RO's roles in ovarian development, female maturation, and adult female fertility.Short narrative: This study employs comprehensive bulk, single cell and single nucleus transcriptome analysis to uncover gene expression signatures of the fetal and adult rete ovarii (RO).
  18. Nat Cell Biol. 2023 Nov 23.
      Gene expression is regulated by multiple epigenetic mechanisms, which are coordinated in development and disease. However, current multiomics methods are frequently limited to one or two modalities at a time, making it challenging to obtain a comprehensive gene regulatory signature. Here, we describe a method-3D genome, RNA, accessibility and methylation sequencing (3DRAM-seq)-that simultaneously interrogates spatial genome organization, chromatin accessibility and DNA methylation genome-wide and at high resolution. We combine 3DRAM-seq with immunoFACS and RNA sequencing in cortical organoids to map the cell-type-specific regulatory landscape of human neural development across multiple epigenetic layers. Finally, we apply a massively parallel reporter assay to profile cell-type-specific enhancer activity in organoids and to functionally assess the role of key transcription factors for human enhancer activation and function. More broadly, 3DRAM-seq can be used to profile the multimodal epigenetic landscape in rare cell types and different tissues.
  19. bioRxiv. 2023 Nov 08. pii: 2023.11.07.566133. [Epub ahead of print]
      Difficult-to-Replicate Sequences (DiToRS) are natural impediments in the human genome that inhibit DNA replication under endogenous replication. Some of the most widely-studied DiToRS are A+T-rich, high "flexibility regions," including long stretches of perfect [AT/TA] microsatellite repeats that have the potential to collapse into hairpin structures when in single-stranded DNA (ssDNA) form and are sites of recurrent structural variation and double-stranded DNA (dsDNA) breaks. Currently, it is unclear how these flexibility regions impact DNA replication, greatly limiting our fundamental understanding of human genome stability. To investigate replication through flexibility regions, we utilized FRET to characterize the effects of the major ssDNA-binding complex, RPA, on the structure of perfect [AT/TA] 25 microsatellite repeats and also re-constituted human lagging strand replication to quantitatively characterize initial encounters of pol δ holoenzymes with A+T-rich DNA template sequences. The results indicate that [AT/TA] 25 sequences adopt hairpin structures that are unwound by RPA and pol δ holoenzymes support dNTP incorporation through the [AT/TA] 25 sequences as well as an A+T-rich, non-structure forming sequence. Furthermore, the extent of dNTP incorporation is dependent on the sequence of the DNA template and the concentration of dNTPs. Importantly, the effects of RPA on the replication of [AT/TA] 25 sequences are dependent on the concentration of dNTPs, whereas the effects of RPA on the replication of an A+T-rich, non-structure forming sequence are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how flexibility regions contribute to genome instability.
  20. Curr Biol. 2023 Nov 10. pii: S0960-9822(23)01506-3. [Epub ahead of print]
      Microtubules in cells consist of functionally diverse subpopulations carrying distinct post-translational modifications (PTMs). Akin to the histone code, the tubulin code regulates a myriad of microtubule functions, ranging from intracellular transport to chromosome segregation. However, how individual PTMs only occur on subsets of microtubules to contribute to microtubule specialization is not well understood. In particular, microtubule detyrosination, the removal of the C-terminal tyrosine on α-tubulin subunits, marks the stable population of microtubules and modifies how microtubules interact with other microtubule-associated proteins to regulate a wide range of cellular processes. Previously, we found that in certain cell types, only ∼30% of microtubules are highly enriched with the detyrosination mark and that detyrosination spans most of the length of a microtubule, often adjacent to a completely tyrosinated microtubule. How the activity of a cytosolic detyrosinase, vasohibin (VASH), leads to only a small subpopulation of highly detyrosinated microtubules is unclear. Here, using quantitative super-resolution microscopy, we visualized nascent microtubule detyrosination events in cells consisting of 1-3 detyrosinated α-tubulin subunits after nocodazole washout. Microtubule detyrosination accumulates slowly and in a dispersed pattern across the microtubule length. By visualizing single molecules of VASH in live cells, we found that VASH engages with microtubules stochastically on a short timescale, suggesting limited removal of tyrosine per interaction, consistent with the super-resolution results. Combining these quantitative imaging results with simulations incorporating parameters from our experiments, we provide evidence for a stochastic model for cells to establish a subset of detyrosinated microtubules via a detyrosination-stabilization feedback mechanism.
    Keywords:  VASH; detyrosination; microtubule dynamics; microtubules; post-translational modifications; single-molecule tracking; super-resolution
  21. Nat Struct Mol Biol. 2023 Nov 23.
      Global changes in transcriptional regulation and RNA metabolism are crucial features of cancer development. However, little is known about the role of the core promoter in defining transcript identity and post-transcriptional fates, a potentially crucial layer of transcriptional regulation in cancer. In this study, we use CAGE-seq analysis to uncover widespread use of dual-initiation promoters in which non-canonical, first-base-cytosine (C) transcription initiation occurs alongside first-base-purine initiation across 59 human cancers and healthy tissues. C-initiation is often followed by a 5' terminal oligopyrimidine (5'TOP) sequence, dramatically increasing the range of genes potentially subjected to 5'TOP-associated post-transcriptional regulation. We show selective, dynamic switching between purine and C-initiation site usage, indicating transcription initiation-level regulation in cancers. We additionally detail global metabolic changes in C-initiation transcripts that mark differentiation status, proliferative capacity, radiosensitivity, and response to irradiation and to PI3K-Akt-mTOR and DNA damage pathway-targeted radiosensitization therapies in colorectal cancer organoids and cancer cell lines and tissues.
  22. Sci Adv. 2023 Nov 24. 9(47): eadg7488
      BMP15 is a conserved regulator of ovarian development and maintenance in vertebrates. In humans, premature ovarian insufficiency is caused by autoimmunity and genetic factors, including mutation of BMP15. The cellular mechanisms underlying ovarian failure caused by BMP15 mutation and immune contributions are not understood. Using zebrafish, we established a causal link between macrophage activation and ovarian failure, which, in zebrafish, causes sex reversal. We define a germline-soma signaling axis that activates macrophages and drives ovarian failure and female-to-male sex reversal. Germline loss of zebrafish Bmp15 impairs oogenesis and initiates this cascade. Single-cell RNA sequencing and genetic analyses implicate ovarian somatic cells that express conserved macrophage-activating ligands as mediators of ovarian failure and sex reversal. Genetic ablation of macrophages or elimination of Csf1Rb ligands, Il34 or Csf1a, delays or blocks premature oocyte loss and sex reversal. The axis identified here provides insight into the cells and pathways governing oocyte and ovary maintenance and potential therapeutic targets to preserve female fertility.
  23. Elife. 2023 Nov 21. pii: RP86507. [Epub ahead of print]12
      In the lesioned zebrafish retina, Müller glia produce multipotent retinal progenitors that generate all retinal neurons, replacing lost cell types. To study the molecular mechanisms linking Müller glia reactivity to progenitor production and neuronal differentiation, we used single-cell RNA sequencing of Müller glia, progenitors and regenerated progeny from uninjured and light-lesioned retinae. We discover an injury-induced Müller glia differentiation trajectory that leads into a cell population with a hybrid identity expressing marker genes of Müller glia and progenitors. A glial self-renewal and a neurogenic trajectory depart from the hybrid cell population. We further observe that neurogenic progenitors progressively differentiate to generate retinal ganglion cells first and bipolar cells last, similar to the events observed during retinal development. Our work provides a comprehensive description of Müller glia and progenitor transcriptional changes and fate decisions in the regenerating retina, which are key to tailor cell differentiation and replacement therapies for retinal dystrophies in humans.
    Keywords:  developmental biology; light lesion; progenitors; regeneration; retina; scRNAseq; zebrafish
  24. J Cell Biochem. 2023 Nov 22.
      The correct assembly of the spindle apparatus directly regulates the precise separation of chromosomes in mouse oocytes, which is crucial for obtaining high-quality oocytes capable of successful fertilization. The localization, assembly, migration, and disassembly of the spindle are regulated by a series of spindle-associated proteins, which exhibit unique expression level variations and specific localization in oocytes. Proteomic analysis revealed that among many representative spindle-associated proteins, the expression level of nucleolar and spindle-associated protein 1 (NUSAP1) significantly increased after meiotic resumption, with a magnitude of change higher than that of other proteins. However, the role of NUSAP1 during oocyte meiosis maturation has not been reported. Here, we report that NUSAP1 is distributed within the cell nucleus during the germinal vesicle (GV) oocytes with non-surrounded nucleolus stage and is not enriched in the nucleus during the GV-surrounded nucleolus stage. Interestingly, NUSAP1 forms distinct granular aggregates near the spindle poles during the prophase of the first meiotic division (Pro-MI), metaphase I, and anaphase I/telophase I stages. Nusap1 depletion leads to chromosome misalignment, increased aneuploidy, and abnormal spindle assembly, particularly a decrease in spindle pole width. Correspondingly, RNA-seq analysis revealed significant suppression of the "establishment of spindle orientation" signaling pathway. Additionally, the attenuation of F-actin in NUSAP1-deficient oocytes may affect the asymmetric division process. Gene ontology analysis of NUSAP1 interactomes, identified through mass spectrometry here, revealed significant enrichment for RNA binding. As an RNA-binding protein, NUSAP1 is likely involved in the regulation of messenger RNA homeostasis by influencing the dynamics of processing (P)-body components. Overall, our results demonstrate the critical importance of precise regulation of NUSAP1 expression levels and protein localization for maintaining mouse oocyte meiosis.
    Keywords:  NUSAP1; meiosis; oocyte; reproduction; spindle
  25. Nat Commun. 2023 Nov 20. 14(1): 7547
      Since adult stem cells are responsible for replenishing tissues throughout life, it is vital to understand how failure to undergo apoptosis can dictate stem cell behavior both intrinsically and non-autonomously. Here, we report that depletion of pro-apoptotic Bax protein bestows hair follicle stem cells with the capacity to eliminate viable neighboring cells by sequestration of TNFα in their membrane. This in turn induces apoptosis in "loser" cells in a contact-dependent manner. Examining the underlying mechanism, we find that Bax loss-of-function competitive phenotype is mediated by the intrinsic activation of NFκB. Notably, winner stem cells differentially respond to TNFα, owing to their elevated expression of TNFR2. Finally, we report that in vivo depletion of Bax results in an increased stem cell pool, accelerating wound-repair and de novo hair follicle regeneration. Collectively, we establish a mechanism of mammalian cell competition, which can have broad therapeutic implications for tissue regeneration and tumorigenesis.
  26. Nat Commun. 2023 Nov 24. 14(1): 7541
      Neurogenesis in the adult mammalian brain relies on the lifelong persistence of quiescent neural stem cell (NSC) reservoirs. Little is known about the mechanisms that lead to the initial establishment of quiescence, the main hallmark of adult stem cells, during development. Here we show that protein aggregates and autophagy machinery components accumulate in developmental radial glia-like NSCs as they enter quiescence and that pharmacological or genetic blockade of autophagy disrupts quiescence acquisition and maintenance. Conversely, increasing autophagy through AMPK/ULK1 activation instructs the acquisition of the quiescent state without affecting BMP signaling, a gatekeeper of NSC quiescence during adulthood. Selective ablation of Atg7, a critical gene for autophagosome formation, in radial glia-like NSCs at early and late postnatal stages compromises the initial acquisition and maintenance of quiescence during the formation of the hippocampal dentate gyrus NSC niche. Therefore, we demonstrate that autophagy is cell-intrinsically required to establish NSC quiescence during hippocampal development. Our results uncover an important role of autophagy in the transition of developmental NSCs into their dormant adult form, paving the way for studies directed at further understanding the mechanisms of stem cell niche formation and maintenance in the mammalian brain.
  27. Mol Cell. 2023 Nov 14. pii: S1097-2765(23)00903-6. [Epub ahead of print]
      Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.
    Keywords:  2CLC; Integrator; MERVL; RNA catabolism; elongation; endogenous retrovirus; non-coding RNA; stem cell; totipotent-like cells; transcription-associated RNA degradation