bims-ginsta Biomed News
on Genome instability
Issue of 2024‒10‒06
thirty-two papers selected by
Jinrong Hu, National University of Singapore



  1. Curr Biol. 2024 Sep 26. pii: S0960-9822(24)01215-6. [Epub ahead of print]
      During cell division, chromosomes build kinetochores that attach to spindle microtubules. Kinetochores usually form at the centromeres, which contain CENP-A nucleosomes. The outer kinetochore, which is the core attachment site for microtubules, is composed of the KMN network (Knl1c, Mis12c, and Ndc80c complexes) and is recruited downstream of CENP-A and its partner CENP-C. In C. elegans oocytes, kinetochores have been suggested to form independently of CENP-A nucleosomes. Yet kinetochore formation requires CENP-C, which acts in parallel to the nucleoporin MEL-28ELYS. Here, we used a combination of RNAi and Degron-based depletion of CENP-A (or downstream CENP-C) to demonstrate that both proteins are in fact responsible for a portion of outer kinetochore assembly during meiosis I and are essential for accurate chromosome segregation. The remaining part requires the coordinated action of KNL-2 (ortholog of human M18BP1) and of the nucleoporin MEL-28ELYS. Accordingly, co-depletion of CENP-A (or CENP-C) and KNL-2M18BP1 (or MEL-28ELYS) prevented outer kinetochore assembly in oocytes during meiosis I. We further found that KNL-2M18BP1 and MEL-28ELYS are interdependent for kinetochore localization. Using engineered mutants, we demonstrated that KNL-2M18BP1 recruits MEL-28ELYS at meiotic kinetochores through a specific N-terminal domain, independently of its canonical CENP-A loading factor activity. Finally, we found that meiosis II outer kinetochore assembly was solely dependent on the canonical CENP-A/CENP-C pathway. Thus, like in most cells, outer kinetochore assembly in C. elegans oocytes depends on centromeric chromatin. However, during meiosis I, an additional KNL-2M18BP1 and MEL-28ELYS pathway acts in a non-redundant manner and in parallel to canonical centromeric chromatin.
    Keywords:  CENP-A; CENP-C; centromere; chromosome segregation; holocentric; kinetochore; microtubule; oocyte meiosis
    DOI:  https://doi.org/10.1016/j.cub.2024.09.004
  2. Science. 2024 Oct 04. 386(6717): eadg7325
      Early embryogenesis is driven by transcription factors (TFs) that first activate the zygotic genome and then specify the lineages constituting the blastocyst. Although the TFs specifying the blastocyst's lineages are well characterized, those playing earlier roles remain poorly defined. Using mouse models of the TF Nr5a2, we show that Nr5a2-/- embryos arrest at the early morula stage and exhibit altered lineage specification, frequent mitotic failure, and substantial chromosome segregation defects. Although NR5A2 plays a minor but measurable role during zygotic genome activation, it predominantly acts as a master regulator at the eight-cell stage, controlling expression of lineage-specifying TFs and genes involved in mitosis, telomere maintenance, and DNA repair. We conclude that NR5A2 coordinates proliferation, genome stability, and lineage specification to ensure correct morula development.
    DOI:  https://doi.org/10.1126/science.adg7325
  3. Nat Commun. 2024 Oct 01. 15(1): 8512
      Dysregulated DNA replication is a cause and a consequence of aneuploidy in cancer, yet the interplay between copy number alterations (CNAs), replication timing (RT) and cell cycle dynamics remain understudied in aneuploid tumors. We developed a probabilistic method, PERT, for simultaneous inference of cell-specific replication and copy number states from single-cell whole genome sequencing (scWGS) data. We used PERT to investigate clone-specific RT and proliferation dynamics in  >50,000 cells obtained from aneuploid and clonally heterogeneous cell lines, xenografts and primary cancers. We observed bidirectional relationships between RT and CNAs, with CNAs affecting X-inactivation producing the largest RT shifts. Additionally, we found that clone-specific S-phase enrichment positively correlated with ground-truth proliferation rates in genomically stable but not unstable cells. Together, these results demonstrate robust computational identification of S-phase cells from scWGS data, and highlight the importance of RT and cell cycle properties in studying the genomic evolution of aneuploid tumors.
    DOI:  https://doi.org/10.1038/s41467-024-52544-7
  4. Nat Struct Mol Biol. 2024 Oct 04.
      Pioneer transcription factors (PTFs) possess the unique capability to access closed chromatin regions and initiate cell fate changes, yet the underlying mechanisms remain elusive. Here, we characterized the single-molecule dynamics of PTFs targeting chromatin in living cells, revealing a notable 'confined target search' mechanism. PTFs such as FOXA1, FOXA2, SOX2, OCT4 and KLF4 sampled chromatin more frequently than non-PTF MYC, alternating between fast free diffusion in the nucleus and slower confined diffusion within mesoscale zones. Super-resolved microscopy showed closed chromatin organized as mesoscale nucleosome-dense domains, confining FOXA2 diffusion locally and enriching its binding. We pinpointed specific histone-interacting disordered regions, distinct from DNA-binding domains, crucial for confined target search kinetics and pioneer activity within closed chromatin. Fusion to other factors enhanced pioneer activity. Kinetic simulations suggested that transient confinement could increase target association rate by shortening search time and binding repeatedly. Our findings illuminate how PTFs recognize and exploit closed chromatin organization to access targets, revealing a pivotal aspect of gene regulation.
    DOI:  https://doi.org/10.1038/s41594-024-01385-5
  5. bioRxiv. 2024 Sep 16. pii: 2024.09.16.613305. [Epub ahead of print]
      Identity-specific interphase chromosome conformation must be re-established each time a cell divides. To understand how interphase folding is inherited, we developed an experimental approach that physically segregates mediators of G1 folding that are intrinsic to mitotic chromosomes from cytoplasmic factors. Proteins essential for nuclear transport, RanGAP1 and Nup93, were degraded in pro-metaphase arrested DLD-1 cells to prevent the establishment of nucleo-cytoplasmic transport during mitotic exit and isolate the decondensing mitotic chromatin of G1 daughter cells from the cytoplasm. Using this approach, we discover a transient folding intermediate entirely driven by chromosome-intrinsic factors. In addition to conventional compartmental segregation, this chromosome-intrinsic folding program leads to prominent genome-scale microcompartmentalization of mitotically bookmarked and cell type-specific cis-regulatory elements. This microcompartment conformation is formed during telophase and subsequently modulated by a second folding program driven by factors inherited through the cytoplasm in G1. This nuclear import-dependent folding program includes cohesin and factors involved in transcription and RNA processing. The combined and inter-dependent action of chromosome-intrinsic and cytoplasmic inherited folding programs determines the interphase chromatin conformation as cells exit mitosis.
    DOI:  https://doi.org/10.1101/2024.09.16.613305
  6. Nat Cell Biol. 2024 Oct 02.
      Aggregates of stem cells can break symmetry and self-organize into embryo-like structures with complex morphologies and gene expression patterns. Mechanisms including reaction-diffusion Turing patterns and cell sorting have been proposed to explain symmetry breaking but distinguishing between these candidate mechanisms of self-organization requires identifying which early asymmetries evolve into subsequent tissue patterns and cell fates. Here we use synthetic 'signal-recording' gene circuits to trace the evolution of signalling patterns in gastruloids, three-dimensional stem cell aggregates that form an anterior-posterior axis and structures resembling the mammalian primitive streak and tailbud. We find that cell sorting rearranges patchy domains of Wnt activity into a single pole that defines the gastruloid anterior-posterior axis. We also trace the emergence of Wnt domains to earlier heterogeneity in Nodal activity even before Wnt activity is detectable. Our study defines a mechanism through which aggregates of stem cells can form a patterning axis even in the absence of external spatial cues.
    DOI:  https://doi.org/10.1038/s41556-024-01521-9
  7. Nat Neurosci. 2024 Sep 30.
      Germinal matrix hemorrhage (GMH) is a devastating neurodevelopmental condition affecting preterm infants, but why blood vessels in this brain region are vulnerable to rupture remains unknown. Here we show that microglia in prenatal mouse and human brain interact with nascent vasculature in an age-dependent manner and that ablation of these cells in mice reduces angiogenesis in the ganglionic eminences, which correspond to the human germinal matrix. Consistent with these findings, single-cell transcriptomics and flow cytometry show that distinct subsets of CD45+ cells from control preterm infants employ diverse signaling mechanisms to promote vascular network formation. In contrast, CD45+ cells from infants with GMH harbor activated neutrophils and monocytes that produce proinflammatory factors, including azurocidin 1, elastase and CXCL16, to disrupt vascular integrity and cause hemorrhage in ganglionic eminences. These results underscore the brain's innate immune cells in region-specific angiogenesis and how aberrant activation of these immune cells promotes GMH in preterm infants.
    DOI:  https://doi.org/10.1038/s41593-024-01769-2
  8. EMBO J. 2024 Sep 30.
      Fibrosis and accumulation of senescent cells are common tissue changes associated with aging. Here, we show that the CDK inhibitor p21 (CDKN1A), known to regulate the cell cycle and the viability of senescent cells, also controls the expression of extracellular matrix (ECM) components in senescent and proliferating cells of the fibrotic lung, in a manner dependent on CDK4 and Rb phosphorylation. p21 knockout protects mice from the induction of lung fibrosis. Moreover, inducible p21 silencing during fibrosis development alleviates disease pathology, decreasing the inflammatory response and ECM accumulation in the lung, and reducing the amount of senescent cells. Furthermore, p21 silencing limits fibrosis progression even when introduced during disease development. These findings show that one common mechanism regulates both cell cycle progression and expression of ECM components, and suggest that targeting p21 might be a new approach for treating age-related fibrotic pathologies.
    Keywords:  CDK4; Cellular Senescence; Extracellular Matrix (ECM); Fibrosis; p21 (CDKN1A)
    DOI:  https://doi.org/10.1038/s44318-024-00246-7
  9. Cell Rep. 2024 Sep 28. pii: S2211-1247(24)01145-8. [Epub ahead of print]43(10): 114794
      Cell division is tightly regulated and requires an expanded energy supply. However, how this energy is generated remains unclear. Here, we establish a correlation between two mitochondrial Ca2+ influx events and ATP production during mitosis. While both events promote ATP production during mitosis, the second event, the Ca2+ influx surge, is substantial. To facilitate this Ca2+ influx surge, the lamin B receptor (LBR) organizes a mitosis-specific endoplasmic reticulum (ER)-mitochondrial contact site (ERMCS), creating a rapid Ca2+ transport pathway. LBR acts as a tether, connecting the ER Ca2+ release channel IP3R with the mitochondrial VDAC2. Depletion of LBR disrupts the Ca2+ influx surge, reduces ATP production, and postpones the metaphase-anaphase transition and subsequent cell division. These findings provide insight into the mechanisms underlying mitotic energy production and supply required for cell proliferation.
    Keywords:  CP: Cell biology; CP: Metabolism; Ca(2+); ER-mitochondrial contact; LBR; VDAC2; cell cycle; cell division; energy generation; metaphase-anaphase transition; mitochondria; mitosis
    DOI:  https://doi.org/10.1016/j.celrep.2024.114794
  10. Curr Biol. 2024 Sep 24. pii: S0960-9822(24)01175-8. [Epub ahead of print]
      Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs.
    Keywords:  Vinculin; Xenopus; actin; adherens junction; barrier function; epithelium; live microscopy; optogenetics; tension; tricellular junctions
    DOI:  https://doi.org/10.1016/j.cub.2024.08.060
  11. STAR Protoc. 2024 Sep 27. pii: S2666-1667(24)00512-4. [Epub ahead of print]5(4): 103347
      Mouse gastrulation entails concomitant changes in cell fate, tissue shape, and embryo size. The use of a reproducible in vitro system is crucial for dissecting the mechanisms that coordinate these events. Here, we present a protocol for generating a 3D culture of epiblast stem cells (3D EpiSCs), which grow as epithelial spheroids mimicking key features of the gastrulating mouse embryonic epiblast. We describe steps for spheroid formation, growth, and passaging, followed by imaging or further downstream analyses. For complete details on the use and execution of this protocol, please refer to Sato et al.1.
    Keywords:  Developmental biology; Organoids; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2024.103347
  12. Mol Cell. 2024 Oct 03. pii: S1097-2765(24)00698-1. [Epub ahead of print]84(19): 3627-3643
      Foundational models of transcriptional regulation involve the assembly of protein complexes at DNA elements associated with specific genes. These assemblies, which can include transcription factors, cofactors, RNA polymerase, and various chromatin regulators, form dynamic spatial compartments that contribute to both gene regulation and local genome architecture. This DNA-protein-centric view has been modified with recent evidence that RNA molecules have important roles to play in gene regulation and genome structure. Here, we discuss evidence that gene regulation by RNA occurs at multiple levels that include assembly of transcriptional complexes and genome compartments, feedback regulation of active genes, silencing of genes, and control of protein kinases. We thus provide an RNA-centric view of transcriptional regulation that must reside alongside the more traditional DNA-protein-centric perspectives on gene regulation and genome architecture.
    Keywords:  RNA; RNA-binding proteins; biomolecular condensates; feedback; gene expression; genome organization; transcription; transcription factors
    DOI:  https://doi.org/10.1016/j.molcel.2024.08.021
  13. Proc Natl Acad Sci U S A. 2024 Oct 08. 121(41): e2408719121
      As ambush-hunting predators that consume large prey after long intervals of fasting, Burmese pythons evolved with unique adaptations for modulating organ structure and function. Among these is cardiac hypertrophy that develops within three days following a meal (Andersen et al., 2005, Secor, 2008), which we previously showed was initiated by circulating growth factors (Riquelme et al., 2011). Postprandial cardiac hypertrophy in pythons also rapidly regresses with subsequent fasting (Secor, 2008); however, the molecular mechanisms that regulate the dynamic cardiac remodeling in pythons during digestion are largely unknown. In this study, we employed a multiomics approach coupled with targeted molecular analyses to examine remodeling of the python ventricular transcriptome and proteome throughout digestion. We found that forkhead box protein O1 (FoxO1) signaling was suppressed prior to hypertrophy development and then activated during regression, which coincided with decreased and then increased expression, respectively, of FoxO1 transcriptional targets involved in proteolysis. To define the molecular mechanistic role of FoxO1 in hypertrophy regression, we used cultured mammalian cardiomyocytes treated with postfed python plasma. Hypertrophy regression both in pythons and in vitro coincided with activation of FoxO1-dependent autophagy; however, the introduction of a FoxO1-specific inhibitor prevented both regression of cell size and autophagy activation. Finally, to determine whether FoxO1 activation could induce regression, we generated an adenovirus expressing a constitutively active FoxO1. FoxO1 activation was sufficient to prevent and reverse postfed plasma-induced hypertrophy, which was partially prevented by autophagy inhibition. Our results indicate that modulation of FoxO1 activity contributes to the dynamic ventricular remodeling in postprandial Burmese pythons.
    Keywords:  Burmese python; FoxO1; autophagy; cardiac hypertrophy; hypertrophy regression
    DOI:  https://doi.org/10.1073/pnas.2408719121
  14. Cell Rep. 2024 Oct 02. pii: S2211-1247(24)01154-9. [Epub ahead of print]43(10): 114803
      Under stress, protein synthesis is attenuated to preserve energy and mitigate challenges to protein homeostasis. Here, we describe, with high temporal resolution, the dynamic landscape of changes in the abundance of proteins synthesized upon stress from transient mitochondrial inner membrane depolarization. This nascent proteome was altered when global translation was attenuated by stress and began to normalize as translation was recovering. This transition was associated with a transient desynchronization of cytosolic and mitochondrial translation and recovery of cytosolic and mitochondrial ribosomal proteins. Further, the elongation factor EEF1A1 was downregulated upon mitochondrial stress, and its silencing mimicked the stress-induced nascent proteome remodeling, including alterations in the nascent respiratory chain proteins. Unexpectedly, the stress-induced alterations in the nascent proteome were independent of physiological protein abundance and turnover. In summary, we provide insights into the physiological and pathological consequences of mitochondrial function and dysfunction.
    Keywords:  CP: Cell biology; CP: Metabolism; EEF1A; EEF1A1; cellular stress; elongation factor; mass spectrometry; mitochondria; nascent chain; protein synthesis; proteomics; translation
    DOI:  https://doi.org/10.1016/j.celrep.2024.114803
  15. Nat Rev Mol Cell Biol. 2024 Oct 02.
      In sexually reproducing organisms, life begins with the fusion of transcriptionally silent gametes, the oocyte and sperm. Although initiation of transcription in the embryo, known as zygotic genome activation (ZGA), is universally required for development, the transcription factors regulating this process are poorly conserved. In this Perspective, we discuss recent insights into the mechanisms of ZGA in totipotent mammalian embryos, namely ZGA regulation by several transcription factors, including by orphan nuclear receptors (OrphNRs) such as the pioneer transcription factor NR5A2, and by factors of the DUX, TPRX and OBOX families. We performed a meta-analysis and compiled a list of pan-ZGA genes, and found that most of these genes are indeed targets of the above transcription factors. Remarkably, more than a third of these ZGA genes appear to be regulated both by OrphNRs such as NR5A2 and by OBOX proteins, whose motifs co-occur in SINE B1 retrotransposable elements, which are enriched near ZGA genes. We propose that ZGA in mice is activated by recruitment of multiple transcription factors to SINE B1 elements that function as enhancers, and discuss a potential relevance of this mechanism to Alu retrotransposable elements in human ZGA.
    DOI:  https://doi.org/10.1038/s41580-024-00772-6
  16. EMBO J. 2024 Oct 04.
      Mitophagy neutralizes mitochondrial damage, thereby preventing cellular dysfunction and apoptosis. Defects in mitophagy have been strongly implicated in age-related neurodegenerative disorders such as Parkinson's and Alzheimer's disease. While mitophagy decreases throughout the lifespan of short-lived model organisms, it remains unknown whether such a decline occurs in the aging mammalian brain-a question of fundamental importance for understanding cell type- and region-specific susceptibility to neurodegeneration. Here, we define the longitudinal dynamics of basal mitophagy and macroautophagy across neuronal and non-neuronal cell types within the intact aging mouse brain in vivo. Quantitative profiling of reporter mouse cohorts from young to geriatric ages reveals cell- and tissue-specific alterations in mitophagy and macroautophagy between distinct subregions and cell populations, including dopaminergic neurons, cerebellar Purkinje cells, astrocytes, microglia and interneurons. We also find that healthy aging is hallmarked by the dynamic accumulation of differentially acidified lysosomes in several neural cell subsets. Our findings argue against any widespread age-related decline in mitophagic activity, instead demonstrating dynamic fluctuations in mitophagy across the aging trajectory, with strong implications for ongoing theragnostic development.
    Keywords:  Aging; Autophagy; Brain; Mitochondria; Mitophagy
    DOI:  https://doi.org/10.1038/s44318-024-00241-y
  17. J Clin Invest. 2024 Oct 03. pii: e180160. [Epub ahead of print]
      Epidermal stem cells control homeostasis and regeneration of skin and hair. In the hair follicle (HF) bulge of mammals, populations of slow-cycling stem cells regenerate the HF during cyclical rounds of anagen (growth), telogen (quiescence), and catagen (regression). Multipotent epidermal cells are also present in the HF above the bulge area, contributing to the formation and maintenance of sebaceous gland and upper and middle portions of the HF. Here, we report that the transcription factor Krox20 is enriched in an epidermal stem cell population located in the upper/ middle HF. Expression analyses and lineage tracing using inducible Krox20-CreERT showed that Krox20-lineage cells migrate out of this HF region and contribute to the formation of bulge in the HF, serving as ancestors of bulge stem cells. In vivo depletion of these cells arrests HF morphogenesis. This study identifies a novel marker for an epidermal stem cell population that is indispensable for hair homeostasis.
    Keywords:  Development; Mouse stem cells; Stem cells
    DOI:  https://doi.org/10.1172/JCI180160
  18. Development. 2024 Oct 01. pii: dev203090. [Epub ahead of print]151(19):
      Cellular plasticity progressively declines with development and differentiation, yet these processes can be experimentally reversed by reprogramming somatic cells to induced pluripotent stem cells (iPSCs) using defined transcription factors. Advances in reprogramming technology over the past 15 years have enabled researchers to study diseases with patient-specific iPSCs, gain fundamental insights into how cell identity is maintained, recapitulate early stages of embryogenesis using various embryo models, and reverse aspects of aging in cultured cells and animals. Here, we review and compare currently available reprogramming approaches, including transcription factor-based methods and small molecule-based approaches, to derive pluripotent cells characteristic of early embryos. Additionally, we discuss our current understanding of mechanisms that resist reprogramming and their role in cell identity maintenance. Finally, we review recent efforts to rejuvenate cells and tissues with reprogramming factors, as well as the application of iPSCs in deriving novel embryo models to study pre-implantation development.
    Keywords:  Cell fate; Epigenetics; Induced pluripotent stem cells; Reprogramming; Small molecules; Transcription factors
    DOI:  https://doi.org/10.1242/dev.203090
  19. Science. 2024 Oct 04. 386(6717): eadl5361
      Kinases are critical regulators of cellular function that are commonly implicated in the mechanisms underlying disease. Most drugs that target kinases are molecules that inhibit their catalytic activity, but here we used chemically induced proximity to convert kinase inhibitors into activators of therapeutic genes. We synthesized bivalent molecules that link ligands of the transcription factor B cell lymphoma 6 (BCL6) to inhibitors of cyclin-dependent kinases (CDKs). These molecules relocalized CDK9 to BCL6-bound DNA and directed phosphorylation of RNA polymerase II. The resulting expression of pro-apoptotic, BCL6-target genes caused killing of diffuse large B cell lymphoma cells and specific ablation of the BCL6-regulated germinal center response. Genomics and proteomics corroborated a gain-of-function mechanism in which global kinase activity was not inhibited but rather redirected. Thus, kinase inhibitors can be used to context-specifically activate transcription.
    DOI:  https://doi.org/10.1126/science.adl5361
  20. Cell. 2024 Sep 25. pii: S0092-8674(24)01019-5. [Epub ahead of print]
      The capability to spatially explore RNA biology in formalin-fixed paraffin-embedded (FFPE) tissues holds transformative potential for histopathology research. Here, we present pathology-compatible deterministic barcoding in tissue (Patho-DBiT) by combining in situ polyadenylation and computational innovation for spatial whole transcriptome sequencing, tailored to probe the diverse RNA species in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for 5 years. Furthermore, genome-wide single-nucleotide RNA variants can be captured to distinguish malignant subclones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis. Single-cell level Patho-DBiT dissects the spatiotemporal cellular dynamics driving tumor clonal architecture and progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to aid in clinical pathology evaluation.
    Keywords:  RNA biology; clinical FFPE tissue; histopathology; microRNA; single-nucleotide RNA variants; spatial omics; spatiotemporal dynamics; splicing isoforms; whole transcriptome
    DOI:  https://doi.org/10.1016/j.cell.2024.09.001
  21. Nat Genet. 2024 Oct 02.
      Aneuploidy, an abnormal chromosome composition, is a major contributor to cancer development and progression and an important determinant of cancer therapeutic responses and clinical outcomes. Despite being recognized as a hallmark of human cancer, the exact role of aneuploidy as a 'driver' of cancer is still largely unknown. Identifying the specific genetic elements that underlie the recurrence of common aneuploidies remains a major challenge of cancer genetics. In this Review, we discuss recurrent aneuploidies and their function as drivers of tumor development. We then delve into the context-dependent identification and functional characterization of the driver genes underlying driver aneuploidies and examine emerging strategies to uncover these driver genes using cancer genomics data and cancer models. Lastly, we explore opportunities for targeting driver aneuploidies in cancer by leveraging the functional consequences of these common genetic alterations.
    DOI:  https://doi.org/10.1038/s41588-024-01916-2
  22. Dev Biol. 2024 Oct 01. pii: S0012-1606(24)00243-4. [Epub ahead of print]
      In the Drosophila testis, developing germ cells are encapsulated by somatic support cells throughout development. Soma-germline interactions are essential for successful spermiogenesis. However, it is still not fully understood what signaling events take place between the soma and the germline. In this study, we found that a Bone Morphogenetic Protein (BMP) ligand, Glass bottom boat (Gbb), secreted from somatic cyst cells (CCs), signals to differentiating germ cells to maintain proper spermiogenesis. Knockdown of Gbb in CCs or the type I BMP receptor Saxophone (Sax) in germ cells leads to a defect in sperm head bundling and decreased fertility. Our Transmission Electron Microscopy (TEM) analyses revealed that the mutant germ cells have aberrant morphology of mitochondria throughout the stages of spermiogenesis and exhibit a defect in nebenkern formation. Elongating spermatids show uncoupled nuclei and elongating mitochondrial derivatives, suggesting that improper mitochondrial development may cause sperm bundling defects. Taken together, we propose a new role of soma-derived BMP signaling, which is essential for spermiogenesis.
    DOI:  https://doi.org/10.1016/j.ydbio.2024.09.016
  23. Nucleic Acids Res. 2024 Oct 01. pii: gkae842. [Epub ahead of print]
      Telomeres protect chromosome ends from DNA damage responses, and their dysfunction triggers genomic alterations like chromosome fusion and rearrangement, which can lead to cellular death. Certain cells, including specific cancer cells, adopt alternative lengthening of telomere (ALT) to counteract dysfunctional telomeres and proliferate indefinitely. While telomere instability and ALT activity are likely major sources of genomic alteration, the patterns and consequences of such changes at the nucleotide level in ALT cells remain unexplored. Here we generated haplotype-resolved genome assemblies for type I ALT mouse embryonic stem cells, facilitated by highly accurate or ultra-long reads and Hi-C reads. High-quality genome revealed ALT-specific complex chromosome end structures and various genomic alterations including over 1000 structural variants (SVs). The unique sequence (mTALT) used as a template for type I ALT telomeres showed traces of being recruited into the genome, with mTALT being replicated with remarkably high accuracy. Subtelomeric regions exhibited distinct characteristics: resistance to the accumulation of SVs and small variants. We genotyped SVs at allele resolution, identifying genes (Rgs6, Dpf3 and Tacc2) crucial for maintaining ALT telomere stability. Our genome assembly-based approach elucidated the unique characteristics of ALT genome, offering insights into the genome evolution of cells surviving telomere-derived crisis.
    DOI:  https://doi.org/10.1093/nar/gkae842
  24. Nature. 2024 Oct 02.
      Ageing impairs the ability of neural stem cells (NSCs) to transition from quiescence to proliferation in the adult mammalian brain. Functional decline of NSCs results in the decreased production of new neurons and defective regeneration following injury during ageing1-4. Several genetic interventions have been found to ameliorate old brain function5-8, but systematic functional testing of genes in old NSCs-and more generally in old cells-has not been done. Here we develop in vitro and in vivo high-throughput CRISPR-Cas9 screening platforms to systematically uncover gene knockouts that boost NSC activation in old mice. Our genome-wide screens in primary cultures of young and old NSCs uncovered more than 300 gene knockouts that specifically restore the activation of old NSCs. The top gene knockouts are involved in cilium organization and glucose import. We also establish a scalable CRISPR-Cas9 screening platform in vivo, which identified 24 gene knockouts that boost NSC activation and the production of new neurons in old brains. Notably, the knockout of Slc2a4, which encodes the GLUT4 glucose transporter, is a top intervention that improves the function of old NSCs. Glucose uptake increases in NSCs during ageing, and transient glucose starvation restores the ability of old NSCs to activate. Thus, an increase in glucose uptake may contribute to the decline in NSC activation with age. Our work provides scalable platforms to systematically identify genetic interventions that boost the function of old NSCs, including in vivo, with important implications for countering regenerative decline during ageing.
    DOI:  https://doi.org/10.1038/s41586-024-07972-2
  25. Nature. 2024 Oct;634(8032): 139-152
    FlyWire Consortium
      The fruit fly Drosophila melanogaster has emerged as a key model organism in neuroscience, in large part due to the concentration of collaboratively generated molecular, genetic and digital resources available for it. Here we complement the approximately 140,000 neuron FlyWire whole-brain connectome1 with a systematic and hierarchical annotation of neuronal classes, cell types and developmental units (hemilineages). Of 8,453 annotated cell types, 3,643 were previously proposed in the partial hemibrain connectome2, and 4,581 are new types, mostly from brain regions outside the hemibrain subvolume. Although nearly all hemibrain neurons could be matched morphologically in FlyWire, about one-third of cell types proposed for the hemibrain could not be reliably reidentified. We therefore propose a new definition of cell type as groups of cells that are each quantitatively more similar to cells in a different brain than to any other cell in the same brain, and we validate this definition through joint analysis of FlyWire and hemibrain connectomes. Further analysis defined simple heuristics for the reliability of connections between brains, revealed broad stereotypy and occasional variability in neuron count and connectivity, and provided evidence for functional homeostasis in the mushroom body through adjustments of the absolute amount of excitatory input while maintaining the excitation/inhibition ratio. Our work defines a consensus cell type atlas for the fly brain and provides both an intellectual framework and open-source toolchain for brain-scale comparative connectomics.
    DOI:  https://doi.org/10.1038/s41586-024-07686-5
  26. Sci Immunol. 2024 Oct 04. 9(100): eado0398
      X chromosome inactivation (XCI) balances X-linked gene dosage between sexes. Unstimulated T cells lack cytological enrichment of X-inactive specific transcript (Xist) RNA and heterochromatic modifications on the inactive X chromosome (Xi), which are involved in maintenance of XCI, and these modifications return to the Xi after stimulation. Here, we examined allele-specific gene expression and epigenomic profiles of the Xi in T cells. We found that the Xi in unstimulated T cells is largely dosage compensated and enriched with the repressive H3K27me3 modification but not the H2AK119-ubiquitin (Ub) mark. Upon T cell stimulation mediated by both CD3 and CD28, the Xi accumulated H2AK119-Ub at gene regions of previous H3K27me3 enrichment. T cell receptor (TCR) engagement, specifically NF-κB signaling downstream of the TCR, was required for Xist RNA localization to the Xi. Disruption of NF-κB signaling in mouse and human T cells using genetic deletion, chemical inhibitors, and patients with immunodeficiencies prevented Xist/XIST RNA accumulation at the Xi and altered X-linked gene expression. Our findings reveal a previously undescribed connection between NF-κB signaling pathways, which affects XCI maintenance in T cells in females.
    DOI:  https://doi.org/10.1126/sciimmunol.ado0398
  27. EMBO J. 2024 Oct 04.
      Alterations in the nuclear envelope are linked to a variety of rare diseases termed laminopathies. A single amino acid substitution at position 12 (A12T) of the human nuclear envelope protein BAF (Barrier to Autointegration Factor) causes Néstor-Guillermo Progeria Syndrome (NGPS). This premature ageing condition leads to growth retardation and severe skeletal defects, but the underlying mechanisms are unknown. Here, we have generated a novel in vivo model for NGPS by modifying the baf-1 locus in C. elegans to mimic the human NGPS mutation. These baf-1(G12T) mutant worms displayed multiple phenotypes related to fertility, lifespan, and stress resistance. Importantly, nuclear morphology deteriorated faster during aging in baf-1(G12T) compared to wild-type animals, recapitulating an important hallmark of cells from progeria patients. Although localization of BAF-1(G12T) was similar to wild-type BAF-1, lamin accumulation at the nuclear envelope was reduced in mutant worms. Tissue-specific chromatin binding and transcriptome analyses showed reduced BAF-1 association in most genes deregulated by the baf-1(G12T) mutation, suggesting that altered BAF chromatin association induces NGPS phenotypes via altered gene expression.
    Keywords:   BANF1 ; Aging; NGPS; Nuclear Lamina; Stress Resistance
    DOI:  https://doi.org/10.1038/s44318-024-00261-8
  28. Elife. 2024 Oct 02. pii: RP90766. [Epub ahead of print]12
      To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here, we focus on the zinc-finger Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1's dissociation from chromatin. Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1's relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1's co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs, and a paradigm for the dual-role of some zinc-finger proteins. The interplay between Sfp1's two modes of transcription regulation remains to be examined.
    Keywords:  S. cerevisiae; chromosomes; gene expression; mRNA decay; mRNA synthesis; transcription backtracking; transcription factor; zinc-finger proteins
    DOI:  https://doi.org/10.7554/eLife.90766
  29. Nat Commun. 2024 Sep 30. 15(1): 8465
      Spermiogenesis, the complex transformation of haploid spermatids into mature spermatozoa, relies on precise spatiotemporal regulation of gene expression at the post-transcriptional level. The mechanisms underlying this critical process remain incompletely understood. Here, we identify centrosomal protein 112 (CEP112) as an essential regulator of mRNA translation during this critical developmental process. Mutations in CEP112 are discovered in oligoasthenoteratospermic patients, and Cep112-deficient male mice recapitulate key phenotypes of human asthenoteratozoospermia. CEP112 localizes to the neck and atypical centrioles of mature sperm and forms RNA granules during spermiogenesis, enriching target mRNAs such as Fsip2, Cfap61, and Cfap74. Through multi-omics analyses and the TRICK reporter assay, we demonstrate that CEP112 orchestrates the translation of target mRNAs. Co-immunoprecipitation and mass spectrometry identify CEP112's interactions with translation-related proteins, including hnRNPA2B1, EEF1A1, and EIF4A1. In vitro, CEP112 undergoes liquid-liquid phase separation, forming condensates that recruit essential proteins and mRNAs. Moreover, variants in patient-derived CEP112 disrupt phase separation and impair translation efficiency. Our results suggest that CEP112 mediates the assembly of RNA granules through liquid-liquid phase separation to control the post-transcriptional expression of fertility-related genes. This study not only clarifies CEP112's role in spermatogenesis but also highlights the role of phase separation in translational regulation, providing insights into male infertility and suggesting potential therapeutic targets.
    DOI:  https://doi.org/10.1038/s41467-024-52705-8
  30. Nature. 2024 Oct 02.
      In mice, intestinal tuft cells have been described as a long-lived, postmitotic cell type. Two distinct subsets have been identified: tuft-1 and tuft-2 (ref. 1). By combining analysis of primary human intestinal resection material and intestinal organoids, we identify four distinct human tuft cell states, two of which overlap with their murine counterparts. We show that tuft cell development depends on the presence of Wnt ligands, and that tuft cell numbers rapidly increase on interleukin-4 (IL-4) and IL-13 exposure, as reported previously in mice2-4. This occurs through proliferation of pre-existing tuft cells, rather than through increased de novo generation from stem cells. Indeed, proliferative tuft cells occur in vivo both in fetal and in adult human intestine. Single mature proliferating tuft cells can form organoids that contain all intestinal epithelial cell types. Unlike stem and progenitor cells, human tuft cells survive irradiation damage and retain the ability to generate all other epithelial cell types. Accordingly, organoids engineered to lack tuft cells fail to recover from radiation-induced damage. Thus, tuft cells represent a damage-induced reserve intestinal stem cell pool in humans.
    DOI:  https://doi.org/10.1038/s41586-024-07952-6
  31. Cell Rep. 2024 Oct 03. pii: S2211-1247(24)01180-X. [Epub ahead of print]43(10): 114829
      Pediatric-type high-grade gliomas frequently harbor gene fusions involving receptor tyrosine kinase genes, including neurotrophic tyrosine kinase receptor (NTRK) fusions. Clinically, these tumors show high initial response rates to tyrosine kinase inhibition but ultimately recur due to the accumulation of additional resistance-conferring mutations. Here, we develop a series of genetically engineered mouse models of treatment-naive and -experienced NTRK1/2/3 fusion-driven gliomas. All tested NTRK fusions are oncogenic in vivo. The NTRK variant, N-terminal fusion partners, and resistance-associated point mutations all influence tumor histology and aggressiveness. Additional tumor suppressor losses greatly enhance tumor aggressiveness. Treatment with TRK kinase inhibitors significantly extends the survival of NTRK fusion-driven glioma mice, but fails to fully eradicate tumors, leading to recurrence upon treatment discontinuation. Finally, we show that ERK activation promotes resistance to TRK kinase inhibition and identify MEK inhibition as a potential combination therapy. These models will be invaluable tools to study therapy resistance of NTRK fusion tumors.
    Keywords:  CP: Cancer; Entrectinib; Larotrectinib; NTRK; RTK; Repotrectinib; Trametinib; gene fusion; mouse models; pediatric glioma; targeted therapy
    DOI:  https://doi.org/10.1016/j.celrep.2024.114829