bims-ginsta Biomed News
on Genome instability
Issue of 2023‒12‒24
29 papers selected by
Jinrong Hu, National University of Singapore

  1. Nature. 2023 Dec 20.
      DNA replication enables genetic inheritance across the kingdoms of life. Replication occurs with a defined temporal order known as the replication timing (RT) programme, leading to organization of the genome into early- or late-replicating regions. RT is cell-type specific, is tightly linked to the three-dimensional nuclear organization of the genome1,2 and is considered an epigenetic fingerprint3. In spite of its importance in maintaining the epigenome4, the developmental regulation of RT in mammals in vivo has not been explored. Here, using single-cell Repli-seq5, we generated genome-wide RT maps of mouse embryos from the zygote to the blastocyst stage. Our data show that RT is initially not well defined but becomes defined progressively from the 4-cell stage, coinciding with strengthening of the A and B compartments. We show that transcription contributes to the precision of the RT programme and that the difference in RT between the A and B compartments depends on RNA polymerase II at zygotic genome activation. Our data indicate that the establishment of nuclear organization precedes the acquisition of defined RT features and primes the partitioning of the genome into early- and late-replicating domains. Our work sheds light on the establishment of the epigenome at the beginning of mammalian development and reveals the organizing principles of genome organization.
  2. J Cell Biol. 2024 Feb 05. pii: e202108070. [Epub ahead of print]223(2):
      Precise chromosome congression and segregation requires the proper assembly of a steady-state metaphase spindle, which is dynamic and maintained by continuous microtubule flux. NuSAP is a microtubule-stabilizing and -bundling protein that promotes chromosome-dependent spindle assembly. However, its function in spindle dynamics remains unclear. Here, we demonstrate that NuSAP regulates the metaphase spindle length control. Mechanistically, NuSAP facilitates kinetochore capture and spindle assembly by promoting Eg5 binding to microtubules. It also prevents excessive microtubule depolymerization through interaction with Kif2A, which reduces Kif2A spindle-pole localization. NuSAP is phosphorylated by Aurora A at Ser-240 during mitosis, and this phosphorylation promotes its interaction with Kif2A on the spindle body and reduces its localization with the spindle poles, thus maintaining proper spindle microtubule flux. NuSAP knockout resulted in the formation of shorter spindles with faster microtubule flux and chromosome misalignment. Taken together, we uncover that NuSAP participates in spindle assembly, dynamics, and metaphase spindle length control through the regulation of microtubule flux and Kif2A localization.
  3. J Cell Biol. 2024 Feb 05. pii: e202304041. [Epub ahead of print]223(2):
      Lattice cells (LCs) in the developing Drosophila retina change shape before attaining final form. Previously, we showed that repeated contraction and expansion of apical cell contacts affect these dynamics. Here, we describe another factor, the assembly of a Rho1-dependent medioapical actomyosin ring formed by nodes linked by filaments that contract the apical cell area. Cell area contraction alternates with relaxation, generating pulsatile changes in cell area that exert force on neighboring LCs. Moreover, Rho1 signaling is sensitive to mechanical changes, becoming active when tension decreases and cells expand, while the negative regulator RhoGAP71E accumulates when tension increases and cells contract. This results in cycles of cell area contraction and relaxation that are reciprocally synchronized between adjacent LCs. Thus, mechanically sensitive Rho1 signaling controls pulsatile medioapical actomyosin contraction and coordinates cell behavior across the epithelium. Disrupting the kinetics of pulsing can lead to developmental errors, suggesting this process controls cell shape and tissue integrity during epithelial morphogenesis of the retina.
  4. Mol Cell. 2023 Dec 07. pii: S1097-2765(23)00965-6. [Epub ahead of print]
      Eukaryotic DNA is packaged into chromatin in the nucleus, restricting the binding of transcription factors (TFs) to their target DNA sites. FOXA1 functions as a pioneer TF to bind condensed chromatin and initiate the opening of local chromatin for gene expression. However, the principles of FOXA1 recruitment and how it subsequently unpacks the condensed chromatin remain elusive. Here, we revealed that FOXA1 intrinsically forms submicron-sized condensates through its N- and C-terminal intrinsically disordered regions (IDRs). Notably, both IDRs enable FOXA1 to dissolve the condensed chromatin. In addition, the DNA-binding capacity of FOXA1 contributes to its ability to both form condensates and dissolve condensed chromatin. Further genome-wide investigation showed that IDRs enable FOXA1 to bind and unpack the condensed chromatin to regulate the proliferation and migration of breast cancer cells. This work provides a principle of how pioneer TFs function to initiate competent chromatin states using their IDRs.
    Keywords:  FOXA1, biomolecular condensates, chromatin, pioneer factor
  5. Curr Biol. 2023 Dec 14. pii: S0960-9822(23)01649-4. [Epub ahead of print]
      Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.
    Keywords:  MPS1; Meiosis; PP2A; REC8; SGO2; aneuploidy; cohesin; human oocytes; maternal aging; reproductive biology
  6. Mol Cell. 2023 Dec 13. pii: S1097-2765(23)00959-0. [Epub ahead of print]
      Genome damage and transcription are intimately linked. Tens to hundreds of thousands of DNA lesions arise in each cell each day, many of which can directly or indirectly impede transcription. Conversely, the process of gene expression is itself a source of endogenous DNA lesions as a result of the susceptibility of single-stranded DNA to damage, conflicts with the DNA replication machinery, and engagement by cells of topoisomerases and base excision repair enzymes to regulate the initiation and progression of gene transcription. Although such processes are tightly regulated and normally accurate, on occasion, they can become abortive and leave behind DNA breaks that can drive genome rearrangements, instability, or cell death.
    Keywords:  DNA repair; R loops; cancer; genome stability; neurodegeneration; replication; transcription
  7. Cell. 2023 Dec 13. pii: S0092-8674(23)01317-X. [Epub ahead of print]
      Deciphering the cell-state transitions underlying immune adaptation across time is fundamental for advancing biology. Empirical in vivo genomic technologies that capture cellular dynamics are currently lacking. We present Zman-seq, a single-cell technology recording transcriptomic dynamics across time by introducing time stamps into circulating immune cells, tracking them in tissues for days. Applying Zman-seq resolved cell-state and molecular trajectories of the dysfunctional immune microenvironment in glioblastoma. Within 24 hours of tumor infiltration, cytotoxic natural killer cells transitioned to a dysfunctional program regulated by TGFB1 signaling. Infiltrating monocytes differentiated into immunosuppressive macrophages, characterized by the upregulation of suppressive myeloid checkpoints Trem2, Il18bp, and Arg1, over 36 to 48 hours. Treatment with an antagonistic anti-TREM2 antibody reshaped the tumor microenvironment by redirecting the monocyte trajectory toward pro-inflammatory macrophages. Zman-seq is a broadly applicable technology, enabling empirical measurements of differentiation trajectories, which can enhance the development of more efficacious immunotherapies.
    Keywords:  cancer; computational biology; dynamics; glioblastoma; immunology; immunotherapy; single-cell biology; systems immunology; temporal transcriptomics; tumor-associated-macrophages
  8. bioRxiv. 2023 Dec 05. pii: 2023.12.04.570024. [Epub ahead of print]
      3D organization of the genome plays a critical role in regulating gene expression. However, it remains unclear how chromatin organization differs among different cell types in the brain. Here we used genome-scale DNA and RNA imaging to investigate 3D-genome organization in transcriptionally distinct cell types in the primary motor cortex of the mouse brain. We uncovered a wide spectrum of differences in the nuclear architecture and 3D-genome organization among different cell types, ranging from the physical size of the cell nucleus to the active-inactive chromatin compartmentalization and radial positioning of chromatin loci within the nucleus. These cell-type-dependent variations in nuclear architecture and chromatin organization exhibited strong correlation with both total transcriptional activity of the cell and transcriptional regulation of cell-type-specific marker genes. Moreover, we found that the methylated-DNA-binding protein MeCP2 regulates transcription in a divergent manner, depending on the nuclear radial positions of chromatin loci, through modulating active-inactive chromatin compartmentalization.
  9. Dev Cell. 2023 Dec 11. pii: S1534-5807(23)00620-2. [Epub ahead of print]
      During morphogenesis, mechanical forces induce large-scale deformations; yet, how forces emerge from cellular contractility and adhesion is unclear. In Drosophila embryos, a tissue-scale wave of actomyosin contractility coupled with adhesion to the surrounding vitelline membrane drives polarized tissue invagination. We show that this process emerges subcellularly from the mechanical coupling between myosin II activation and sequential adhesion/de-adhesion to the vitelline membrane. At the wavefront, integrin clusters anchor the actin cortex to the vitelline membrane and promote activation of myosin II, which in turn enhances adhesion in a positive feedback. Following cell detachment, cortex contraction and advective flow amplify myosin II. Prolonged contact with the vitelline membrane prolongs the integrin-myosin II feedback, increases integrin adhesion, and thus slows down cell detachment and wave propagation. The angle of cell detachment depends on adhesion strength and sets the tensile forces required for detachment. Thus, we document how the interplay between subcellular mechanochemical feedback and geometry drives tissue morphogenesis.
    Keywords:  Drosophila; actomyosin contractility; embryo development; morphogenesis; substrate adhesion
  10. Curr Biol. 2023 Dec 15. pii: S0960-9822(23)01655-X. [Epub ahead of print]
      Metazoan development relies on the formation and remodeling of cell-cell contacts. Dynamic reorganization of adhesion receptors and the actomyosin cell cortex in space and time plays a central role in cell-cell contact formation and maturation. Nevertheless, how this process is mechanistically achieved when new contacts are formed remains unclear. Here, by building a biomimetic assay composed of progenitor cells adhering to supported lipid bilayers functionalized with E-cadherin ectodomains, we show that cortical F-actin flows, driven by the depletion of myosin-2 at the cell contact center, mediate the dynamic reorganization of adhesion receptors and cell cortex at the contact. E-cadherin-dependent downregulation of the small GTPase RhoA at the forming contact leads to both a depletion of myosin-2 and a decrease of F-actin at the contact center. At the contact rim, in contrast, myosin-2 becomes enriched by the retraction of bleb-like protrusions, resulting in a cortical tension gradient from the contact rim to its center. This tension gradient, in turn, triggers centrifugal F-actin flows, leading to further accumulation of F-actin at the contact rim and the progressive redistribution of E-cadherin from the contact center to the rim. Eventually, this combination of actomyosin downregulation and flows at the contact determines the characteristic molecular organization, with E-cadherin and F-actin accumulating at the contact rim, where they are needed to mechanically link the contractile cortices of the adhering cells.
    Keywords:  actomyosin; cadherin; cell adhesion; contact patterning; cortical flow; cortical tension; cytoskeleton; supported lipid bilayers
  11. Trends Cell Biol. 2023 Dec 21. pii: S0962-8924(23)00240-4. [Epub ahead of print]
      Mitochondria are activated during stem cell differentiation. Recently, Wang et al. found that mechanical stimulation from tissue surrounding differentiating germ cells in the female fly ovary is necessary to sustain intracellular calcium levels, promoting mitochondrial activity. This suggests a molecular link between cell mechanics and developmental metabolic transitions in eukaryotes.
  12. J Clin Invest. 2023 Dec 21. pii: e172116. [Epub ahead of print]
      Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations in pulmonary mesenchymal cells resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of LAM patients develops pulmonary vascular remodeling and pulmonary hypertension. Little, however, is known regarding how LAM cells communicate with endothelial cells (ECs) to trigger vascular remodeling. In end-stage LAM lung explants, we identified endothelial cell dysfunction characterized by increased proliferation, migration, defective angiogenesis, and dysmorphic endothelial tube network formation. To model LAM disease, we utilized an mTORC1 gain-of-function mouse model with a Tsc2 knock-out (Tsc2KO) specific to lung mesenchyme (Tbx4LME-CreTsc2fl/fl), similar to the mesenchyme specific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from Tbx4LME-CreTsc2fl/fl mice exhibited marked transcriptomic changes despite absence of morphological changes to the distal lung microvasculature. In contrast, 1 year old Tbx4LME-CreTsc2fl/fl mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. Single cell RNA-sequencing of 1 year old mouse lung identified paracrine ligands originating from Tsc2KO mesenchyme which can signal through receptors in arterial ECs. These ECs had transcriptionally altered genes including those in pathways associated with blood vessel remodeling. The proposed pathophysiologic mesenchymal ligand/ EC receptor crosstalk highlights the importance of an altered mesenchymal-EC axis in LAM and other hyperactive mTORC1-driven diseases. Since ECs in LAM patients and in Tbx4LME-CreTsc2fl/fl mice do not harbor TSC2 mutations, our study demonstrates that constitutively active mTORC1 lung mesenchymal cells orchestrate dysfunctional EC responses which contribute to pulmonary vascular remodeling.
    Keywords:  Cell Biology; Endothelial cells; Mouse models; Vascular Biology
  13. Cell Rep. 2023 Dec 18. pii: S2211-1247(23)01605-4. [Epub ahead of print]43(1): 113593
      Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that, upon glucose withdrawal, the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and control gene expression during stress.
    Keywords:  CP: Cell biology; DEAD-box ATPase Dbp5; Nab2; RNA; condensation; glucose stress; mRNA export
  14. Mol Cell. 2023 Dec 11. pii: S1097-2765(23)00969-3. [Epub ahead of print]
      Cellular homeostasis is constantly challenged by a myriad of extrinsic and intrinsic stressors. To mitigate the stress-induced damage, cells activate transient survival programs. The heat shock response (HSR) is an evolutionarily well-conserved survival program that is activated in response to proteotoxic stress. The HSR encompasses a dual regulation of transcription, characterized by rapid activation of genes encoding molecular chaperones and concomitant global attenuation of non-chaperone genes. Recent genome-wide approaches have delineated the molecular depth of stress-induced transcriptional reprogramming. The dramatic rewiring of gene and enhancer networks is driven by key transcription factors, including heat shock factors (HSFs), that together with chromatin-modifying enzymes remodel the 3D chromatin architecture, determining the selection of either gene activation or repression. Here, we highlight the current advancements of molecular mechanisms driving transcriptional reprogramming during acute heat stress. We also discuss the emerging implications of HSF-mediated stress signaling in the context of physiological and pathological conditions.
    Keywords:  HSF; HSR; PTM; Pol II; RNA polymerase II; chromatin; enhancer; heat shock factor; heat shock response; post-translational modification; proteostasis; transcription; transcription factor
  15. Nat Metab. 2023 Dec;5(12): 2094-2110
      The thyroid functions as an apex endocrine organ that controls growth, differentiation and metabolism1, and thyroid diseases comprise the most common endocrine disorders2. Nevertheless, high-resolution views of the cellular composition and signals that govern the thyroid have been lacking3,4. Here, we show that Notch signalling controls homeostasis and thermoregulation in adult mammals through a mitochondria-based mechanism in a subset of thyrocytes. We discover two thyrocyte subtypes in mouse and human thyroids, identified in single-cell analyses by different levels of metabolic activity and Notch signalling. Therapeutic antibody blockade of Notch in adult mice inhibits a thyrocyte-specific transcriptional program and induces thyrocyte defects due to decreased mitochondrial activity and ROS production. Thus, disrupting Notch signalling in adult mice causes hypothyroidism, characterized by reduced levels of circulating thyroid hormone and dysregulation of whole-body thermoregulation. Inducible genetic deletion of Notch1 and 2 in thyrocytes phenocopies this antibody-induced hypothyroidism, establishing a direct role for Notch in adult murine thyrocytes. We confirm that hypothyroidism is enriched in children with Alagille syndrome, a genetic disorder marked by Notch mutations, suggesting that these findings translate to humans.
  16. bioRxiv. 2023 Dec 04. pii: 2023.12.03.569799. [Epub ahead of print]
      To survive daily damage, the formation of actomyosin ring at the wound periphery is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. Here, we find that rapid recruitment of all three Drosophila calcium responding and phospholipid binding Annexin proteins (AnxB9, AnxB10, AnxB11) to distinct regions around the wound are regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, we find that reduced extracellular calcium and depletion of intracellular calcium affect cell wound repair differently, despite these two conditions exhibiting similar GCaMP signals. Thus, our results suggest that, in addition to initiating repair events, both the quantity and sources of calcium influx are important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair.Summary: Cells have rapid and robust repair systems to survive daily damage. This study shows that calcium influx regulates the three distinct Drosophila Annexin recruitment patterns to the cell wound in order to organize an actomyosin ring for efficient wound closure.
  17. Mol Cell. 2023 Dec 21. pii: S1097-2765(23)00970-X. [Epub ahead of print]83(24): 4509-4523.e11
      The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.
    Keywords:  3′ UTR; CDS exon length; RNA-binding proteins; TIAL1; TIS granules; TIS11B; condensates; cytoplasmic organization; endoplasmic reticulum; gene architecture; mRNA length; mRNA localization; spatial regulation of protein synthesis; translation environment
  18. Yeast. 2023 Dec 21.
      Eukaryotic genes must be condensed into chromatin while remaining accessible to the transcriptional machinery to support gene expression. Among the three eukaryotic RNA polymerases (RNAP), RNAPII is unique, partly because of the C-terminal domain (CTD) of its largest subunit, Rpb1. Rpb1 CTD can be extensively modified during the transcription cycle, allowing for the co-transcriptional recruitment of specific interacting proteins. These include chromatin remodeling factors that control the opening or closing of chromatin. How the CTD-less RNAPI and RNAPIII deal with chromatin at rRNA and tRNA genes is less understood. Here, we review recent advances in our understanding of how the chromatin at tRNA genes and rRNA genes can be remodeled in response to environmental cues in yeast, with a particular focus on the role of local RNAPII transcription in recruiting chromatin remodelers at these loci. In fission yeast, RNAPII transcription at tRNA genes is important to re-establish a chromatin environment permissive to tRNA transcription, which supports growth from stationary phase. In contrast, local RNAPII transcription at rRNA genes correlates with the closing of the chromatin in starvation in budding and fission yeast, suggesting a role in establishing silent chromatin. These opposite roles might support a general model where RNAPII transcription recruits chromatin remodelers to tRNA and rRNA genes to promote the closing and reopening of chromatin in response to the environment.
    Keywords:  RNA polymerase II; chromatin; histone; transcription
  19. Mol Cell. 2023 Dec 09. pii: S1097-2765(23)00973-5. [Epub ahead of print]
      Cellular homeostasis is continuously challenged by environmental cues and cellular stress conditions. In their defense, cells need to mount appropriate stress responses that, dependent on the cellular context, signaling intensity, and duration, may have diverse outcomes. The stress- and mitogen-activated protein kinase (SAPK/MAPK) system consists of well-characterized signaling cascades that sense and transduce an array of different stress stimuli into biological responses. However, the physical and chemical nature of stress signals and how these are sensed by individual upstream MAP kinase kinase kinases (MAP3Ks) remain largely ambiguous. Here, we review the existing knowledge of how individual members of the large and diverse group of MAP3Ks sense specific stress signals through largely non-redundant mechanisms. We emphasize the large knowledge gaps in assigning function and stress signals for individual MAP3K family members and touch on the potential of targeting this class of proteins for clinical benefit.
    Keywords:  JNK; MAP3K; cellular stress response; p38
  20. bioRxiv. 2023 Dec 09. pii: 2023.12.08.570828. [Epub ahead of print]
      The eukaryotic genome, first packed into nucleosomes of about 150 bp around the histone core, is organized into euchromatin and heterochromatin, corresponding to the A and B compartments, respectively. Here, we asked if individual nucleosomes in vivo know where to go. That is, do mono-nucleosomes by themselves contain A/B compartment information, associated with transcription activity, in their biophysical properties? We purified native mono-nucleosomes to high monodispersity and used physiological concentrations of biological polyamines to determine their condensability. The chromosomal regions known to partition into A compartments have low condensability and vice versa. In silico chromatin polymer simulations using condensability as the only input showed that biophysical information needed to form compartments is all contained in single native nucleosomes and no other factors are needed. Condensability is also strongly anticorrelated with gene expression, and especially so near the promoter region and in a cell type dependent manner. Therefore, individual nucleosomes in the promoter know whether the gene is on or off, and that information is contained in their biophysical properties. Comparison with genetic and epigenetic features suggest that nucleosome condensability is a very meaningful axis onto which to project the high dimensional cellular chromatin state. Analysis of condensability using various condensing agents including those that are protein-based suggests that genome organization principle encoded into individual nucleosomes is electrostatic in nature. Polyamine depletion in mouse T cells, by either knocking out ornithine decarboxylase (ODC) or inhibiting ODC, results in hyperpolarized condensability, suggesting that when cells cannot rely on polyamines to translate biophysical properties of nucleosomes to control gene expression and 3D genome organization, they accentuate condensability contrast, which may explain dysfunction known to occur with polyamine deficiency.
  21. Dev Cell. 2023 Dec 08. pii: S1534-5807(23)00621-4. [Epub ahead of print]
      Autophagy is a conserved cellular degradation process. While autophagy-related proteins were shown to influence the signaling and trafficking of some receptor tyrosine kinases, the relevance of this during cancer development is unclear. Here, we identify a role for autophagy in regulating platelet-derived growth factor receptor alpha (PDGFRA) signaling and levels. We find that PDGFRA can be targeted for autophagic degradation through the activity of the autophagy cargo receptor p62. As a result, short-term autophagy inhibition leads to elevated levels of PDGFRA but an unexpected defect in PDGFA-mediated signaling due to perturbed receptor trafficking. Defective PDGFRA signaling led to its reduced levels during prolonged autophagy inhibition, suggesting a mechanism of adaptation. Importantly, PDGFA-driven gliomagenesis in mice was disrupted when autophagy was inhibited in a manner dependent on Pten status, thus highlighting a genotype-specific role for autophagy during tumorigenesis. In summary, our data provide a mechanism by which cells require autophagy to drive tumor formation.
    Keywords:  PDGFRA; PTEN; RTK; autophagy; cancer; endocytosis; glioblastoma; signaling
  22. Cell Rep. 2023 Dec 19. pii: S2211-1247(23)01615-7. [Epub ahead of print]43(1): 113603
      The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase with important roles in many cellular processes as well as in cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. How these dimers relate to higher-order EGFR oligomers seen in cell membranes, however, remains unclear. Here, we used single-particle tracking (SPT) and Förster resonance energy transfer imaging to examine how each domain of EGFR contributes to receptor oligomerization and the rate of receptor diffusion in the cell membrane. Although the extracellular region of EGFR is sufficient to drive receptor dimerization, we find that the EGF-induced EGFR slowdown seen by SPT requires higher-order oligomerization-mediated in part by the intracellular tyrosine kinase domain when it adopts an active conformation. Our data thus provide important insight into the interactions required for higher-order EGFR assemblies involved in EGF signaling.
    Keywords:  CP: Molecular biology; cancer; clustering; dimerization; epidermal growth factor receptor; fluorescence; imaging; receptor tyrosine kinase; single particle tracking; transmembrane signaling; tyrosine kinase
  23. bioRxiv. 2023 Dec 07. pii: 2023.12.06.570417. [Epub ahead of print]
      The 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 patient brain tissue shows that large-scale transcriptional changes occur in patient cerebellum that correlate with expression level and 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 ROS 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.
  24. bioRxiv. 2023 Dec 07. pii: 2023.12.05.570186. [Epub ahead of print]
      Stem cells have lower facultative heterochromatin as defined by trimethylation of histone H3 lysine 27 (H3K27me3) compared to differentiated cells, however, the underlying mechanism for this observation has been unknown. Because H3K27me3 levels are diluted two-fold in every round of replication and then restored through the rest of the cycle, we reasoned that the cell cycle length could determine the time available for setting total H3K27me3 levels. Here, we demonstrate that a fast cell cycle sets low levels of H3K27me3 in serum-grown murine embryonic stem cells (mESCs). Extending the G1 phase leads to an increase in global H3K27me3 in mESCs due to the formation of de novo Polycomb domains, and the length of the G1/S block correlates with the extent of gain in H3K27me3, arguing that levels of the modification depend on the time available between successive rounds of replication. Similarly, accelerating the cell cycle in HEK293 cells decreases H3K27me3 levels. Finally, we applied this principle in tumor cells that, due to the dominant negative effect of the sub-stoichiometric H3K27M mutant, have reduced H3K27me3. Here, extending G1 using Palbociclib increased H3K27me3 levels, pointing to an unexpected means to rescue the effect of oncohistones. Our results suggest cell cycle length as a universal mechanism to modulate heterochromatin formation and, thus, cellular identity.
  25. Mol Cell. 2023 Dec 11. pii: S1097-2765(23)00968-1. [Epub ahead of print]
      Autophagy, an important quality control and recycling process vital for cellular homeostasis, is tightly regulated. The mTORC1 signaling pathway regulates autophagy under conditions of nutrient availability and scarcity. However, how mTORC1 activity is fine-tuned during nutrient availability to allow basal autophagy is unclear. Here, we report that the WD-domain repeat protein MORG1 facilitates basal constitutive autophagy by inhibiting mTORC1 signaling through Rag GTPases. Mechanistically, MORG1 interacts with active Rag GTPase complex inhibiting the Rag GTPase-mediated recruitment of mTORC1 to the lysosome. MORG1 depletion in HeLa cells increases mTORC1 activity and decreases autophagy. The autophagy receptor p62/SQSTM1 binds to MORG1, but MORG1 is not an autophagy substrate. However, p62/SQSTM1 binding to MORG1 upon re-addition of amino acids following amino acid's depletion precludes MORG1 from inhibiting the Rag GTPases, allowing mTORC1 activation. MORG1 depletion increases cell proliferation and migration. Low expression of MORG1 correlates with poor survival in several important cancers.
    Keywords:  MORG1; Rag GTPases; WD-domain repeat protein; autophagy; mTORC1; p62/SQSTM1
  26. Nature. 2024 Jan;625(7993): 35-36
    Keywords:  Cell biology; Immunology; Physiology
  27. Dev Cell. 2023 Dec 19. pii: S1534-5807(23)00622-6. [Epub ahead of print]
      Organ size is controlled by numerous factors including mechanical forces, which are mediated in part by the Hippo pathway. In growing Drosophila epithelial tissues, cytoskeletal tension influences Hippo signaling by modulating the localization of key pathway proteins to different apical domains. Here, we discovered a Hippo signaling hub at basal spot junctions, which form at the basal-most point of the lateral membranes and resemble adherens junctions in protein composition. Basal spot junctions recruit the central kinase Warts via Ajuba and E-cadherin, which prevent Warts activation by segregating it from upstream Hippo pathway proteins. Basal spot junctions are prominent when tissues undergo morphogenesis and are highly sensitive to fluctuations in cytoskeletal tension. They are distinct from focal adhesions, but the latter profoundly influences basal spot junction abundance by modulating the basal-medial actomyosin network and tension experienced by spot junctions. Thus, basal spot junctions couple morphogenetic forces to Hippo pathway activity and organ growth.
    Keywords:  E-cadherin; Hippo signaling; Warts kinase; basal spot junctions; cell adhesion; focal adhesions; growth control; mechanobiology; morphogenesis; myosin
  28. Nat Aging. 2023 Dec 15.
      Over the past decade, there has been a dramatic increase in efforts to ameliorate aging and the diseases it causes, with transient expression of nuclear reprogramming factors recently emerging as an intriguing approach. Expression of these factors, either systemically or in a tissue-specific manner, has been shown to combat age-related deterioration in mouse and human model systems at the cellular, tissue and organismal level. Here we discuss the current state of epigenetic rejuvenation strategies via partial reprogramming in both mouse and human models. For each classical reprogramming factor, we provide a brief description of its contribution to reprogramming and discuss additional factors or chemical strategies. We discuss what is known regarding chromatin remodeling and the molecular dynamics underlying rejuvenation, and, finally, we consider strategies to improve the practical uses of epigenetic reprogramming to treat aging and age-related diseases, focusing on the open questions and remaining challenges in this emerging field.
  29. Cell Metab. 2023 Dec 09. pii: S1550-4131(23)00443-6. [Epub ahead of print]
      A major hypothesis for the etiology of type 1 diabetes (T1D) postulates initiation by viral infection, leading to double-stranded RNA (dsRNA)-mediated interferon response and inflammation; however, a causal virus has not been identified. Here, we use a mouse model, corroborated with human islet data, to demonstrate that endogenous dsRNA in beta cells can lead to a diabetogenic immune response, thus identifying a virus-independent mechanism for T1D initiation. We found that disruption of the RNA editing enzyme adenosine deaminases acting on RNA (ADAR) in beta cells triggers a massive interferon response, islet inflammation, and beta cell failure and destruction, with features bearing striking similarity to early-stage human T1D. Glycolysis via calcium enhances the interferon response, suggesting an actionable vicious cycle of inflammation and increased beta cell workload.
    Keywords:  RNA editing; beta cells; interferon response; islet inflammation; metabolic stress; type 1 diabetes