scholarly journals Nucleophagy contributes to genome stability though TOP2cc and nucleolar components degradation

2021 ◽  
Author(s):  
Gabriel Muciño-Hernández ◽  
Adán Oswaldo Guerrero Cárdenas ◽  
Horacio Merchant-Larios ◽  
Susana Castro-Obregón
Keyword(s):  
Mammalian Cells ◽  
Genome Stability ◽  
Nuclear Architecture ◽  
Nuclear Dynamics ◽  
Nucleolar Stress ◽  
Primary Mouse ◽  
Nuclear Stability ◽  
Type Ii Dna Topoisomerases ◽  
Nuclear Alterations ◽  
Autophagic Proteins

ABSTRACTThe nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal, healthy cells maintain genome stability avoiding accumulation of nuclear damaged material is less understood. Here we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after Etoposide-induced DNA Double-Stranded Breaks. These nuclear buds and micronuclei co-localize with autophagic proteins BECN1 and LC3 and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and Type II DNA Topoisomerases (TOP2A and TOP2B), or nucleolar protein Fibrillarin, implying they are also targets of nucleophagy. We propose that a basal nucleophagy contributes to genome and nuclear stability and also in response to DNA damage and nucleolar stress.

Nucleolar Stress and p53 Mediated Erythroid Failure in Diamond Blackfan Anemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 42-42
Author(s):  
Colin Sieff ◽  
Harvey F. Lodish
Keyword(s):  
Cell Cycle ◽  
Ribosomal Protein ◽  
Cell Cycle Arrest ◽  
Ribosomal Rna ◽  
Mammalian Cells ◽  
Pcr Analysis ◽  
Erythroid Cells ◽  
Cycle Arrest ◽  
Nucleolar Stress ◽  
Primary Mouse

Abstract The discovery that several ribosomal protein genes can be mutated in DBA suggests that ribosomal protein gene mutations may account for many or all cases of DBA, and focuses attention on the ribosome. While experiments in yeast and mammalian cells show that RPS19 depletion or mutation leads to a block in ribosomal RNA biosynthesis, this result does not explain why erythropoiesis is so severely affected in DBA. We hypothesize that during fetal development immature erythroid cells proliferate more rapidly than other lineages and therefore require very high ribosome synthetic rates to generate sufficient capacity for translation of erythroid specific transcripts that must take place before these unique cells enucleate. To test this kinetic hypothesis we measured RNA biogenesis in primary mouse fetal liver cells and reported previously that during the first 24 hours cell number increases 3–4 fold while, remarkably, there is a 6-fold increase in RNA content during the same period, suggesting that the cells accumulate an excess of ribosomal RNA (80% of measured RNA) during early erythropoiesis. Retrovirus infected siRNA RPS19 knockdown cells show reduced proliferation of FACS sorted GFP positive cells at 48 hours. Although the cell yield is reduced, the differentiation pattern of the surviving GFP positive cells is similar to that of the controls. While quantitative RT-PCR analysis shows that RPS19 mRNA is rapidly depleted, Western analysis during this time course does not show a deficiency of RPS19 protein. This suggests strongly that the proliferative defect is not due to insufficiency of RPS19 protein, and is more likely due nucleolar stress induced by the block in ribosome biogenesis. Molecular consequences could lead to redistribution of cell cycle proteins normally resident in the nucleolus with consequent p53 mediated cell cycle arrest and or apoptosis. To test this hypothesis we used a culture system that allows expansion without differentiation of immature cells in SCF, EPO, IGF-1 and dexamethasone. Under these conditions proliferation of siRNA expressing precursors is reduced with an increased proportion arrested in G0/G1 in the knockdown cells. Furthermore, p53 is increased in the knockdown cells. Taken together, these data suggest that RPS19 insufficient cells undergo a nucleolar stress response and erythroid cells proliferate poorly because of p53 mediated cell cycle arrest and apoptosis.

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

scholarly journals Alternative oxidase encoded by sequence-optimized and chemically-modified RNA transfected into mammalian cells is catalytically active

Gene Therapy ◽  
2021 ◽  
Author(s):  
Luca Giordano ◽  
Manish K. Aneja ◽  
Natascha Sommer ◽  
Nasim Alebrahimdehkordi ◽  
Alireza Seraji ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Alternative Oxidase ◽  
Chemically Modified ◽  
Lung Carcinoma Cells ◽  
Primary Mouse ◽  
Catalytically Active ◽  
Human Lung Carcinoma ◽  
Pulmonary Arterial ◽  
Metabolic Stresses ◽  
Mitochondrial Respiratory

AbstractPlants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders.

scholarly journals Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos using CAS-LiveFISH

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yongtao Geng ◽  
Alexandros Pertsinidis
Keyword(s):  
Mammalian Cells ◽  
Target Gene ◽  
Nuclear Architecture ◽  
Live Cells ◽  
Transcription Activator ◽  
Distal Enhancer ◽  
Chromatin Dynamics ◽  
Guide Rnas ◽  
Early Mouse

AbstractVisualizing the 4D genome in live cells is essential for understanding its regulation. Programmable DNA-binding probes, such as fluorescent clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector (TALE) proteins have recently emerged as powerful tools for imaging specific genomic loci in live cells. However, many such systems rely on genetically-encoded components, often requiring multiple constructs that each must be separately optimized, thus limiting their use. Here we develop efficient and versatile systems, based on in vitro transcribed single-guide-RNAs (sgRNAs) and fluorescently-tagged recombinant, catalytically-inactivated Cas9 (dCas9) proteins. Controlled cell delivery of pre-assembled dCas9-sgRNA ribonucleoprotein (RNP) complexes enables robust genomic imaging in live cells and in early mouse embryos. We further demonstrate multiplex tagging of up to 3 genes, tracking detailed movements of chromatin segments and imaging spatial relationships between a distal enhancer and a target gene, with nanometer resolution in live cells. This simple and effective approach should facilitate visualizing chromatin dynamics and nuclear architecture in various living systems.

scholarly journals The Protective Role of Dormant Origins in Response to Replicative Stress

2018 ◽  
Vol 19 (11) ◽  
pp. 3569 ◽  
Author(s):  
Lilas Courtot ◽  
Jean-Sébastien Hoffmann ◽  
Valérie Bergoglio
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Replication Timing ◽  
Protective Role ◽  
Entire Genome ◽  
Replicative Stress ◽  
A Cell ◽  
The Many

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20–30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or “dormant” origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress.

scholarly journals Arp2/3 and Unc45 maintain heterochromatin stability in Drosophila polytene chromosomes

2019 ◽  
Author(s):  
George Dialynas ◽  
Laetitia Delabaere ◽  
Irene Chiolo
Keyword(s):  
Actin Filaments ◽  
Genome Stability ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Nuclear Periphery ◽  
Polytene Chromosomes ◽  
Nuclear Actin ◽  
Nuclear Dynamics ◽  
Ectopic Recombination ◽  
Repair Pathways

AbstractRepairing DNA double-strand breaks (DSBs) is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination. InDrosophilaKc cells, accurate homologous recombination (HR) repair of heterochromatic DSBs relies on the relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. This movement is driven by Arp2/3-dependent nuclear actin filaments and myosins’ ability to walk along them. Conserved mechanisms enable the relocalization of heterochromatic DSBs in mouse cells, and their defects lead to massive ectopic recombination in heterochromatin and chromosome rearrangements. InDrosophilapolytene chromosomes, extensive DNA movement is blocked by a stiff structure of chromosome bundles. Repair pathways in this context are poorly characterized, and whether heterochromatic DSBs relocalize in these cells is unknown. Here, we show that damage in heterochromatin results in relaxation of the heterochromatic chromocenter, consistent with a dynamic response in this structure. Arp2/3, the Arp2/3 activator Scar, and the myosin activator Unc45, are required for heterochromatin stability in polytene cells, suggesting that relocalization enables heterochromatin repair in this tissue. Together, these studies reveal critical roles for actin polymerization and myosin motors in heterochromatin repair and genome stability across different organisms and tissue types.Impact StatementHeterochromatin relies on dedicated pathways for ‘safe’ recombinational repair. In mouse and fly cultured cells, DNA repair requires the movement of repair sites away from the heterochromatin ‘domain’vianuclear actin filaments and myosins. Here, we explore the importance of these pathways inDrosophilasalivary gland cells, which feature a stiff bundle of endoreduplicated polytene chromosomes. Repair pathways in polytene chromosomes are largely obscure and how nuclear dynamics operate in this context is unknown. We show that heterochromatin relaxes in response to damage, and relocalization pathways are necessary for repair and stability of heterochromatic sequences. This deepens our understanding of repair mechanisms in polytenes, revealing unexpected dynamics. It also provides a first understanding of nuclear dynamics responding to replication damage or rDNA breaks, providing a new understanding of the importance of the nucleoskeleton in genome stability. We expect these discoveries to shed light on tumorigenic processes, including therapy-induced cancer relapses.

scholarly journals Control of the DNA Methylation System Component MBD2 by Protein Arginine Methylation

2006 ◽  
Vol 26 (19) ◽  
pp. 7224-7235 ◽  
Author(s):  
Choon Ping Tan ◽  
Sara Nakielny
Keyword(s):  
Dna Methylation ◽  
Complex Formation ◽  
Histone Deacetylases ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Epigenetic Modification ◽  
Arginine Methylation ◽  
System Component ◽  
Transcription Repression ◽  
Mbd Proteins

ABSTRACT DNA methylation is vital for proper chromatin structure and function in mammalian cells. Genetic removal of the enzymes that catalyze DNA methylation results in defective imprinting, transposon silencing, X chromosome dosage compensation, and genome stability. This epigenetic modification is interpreted by methyl-DNA binding domain (MBD) proteins. MBD proteins respond to methylated DNA by recruiting histone deacetylases (HDAC) and other transcription repression factors to the chromatin. The MBD2 protein is dispensable for animal viability, but it is implicated in the genesis of colon tumors. Here we report that the MBD2 protein is controlled by arginine methylation. We identify the protein arginine methyltransferase enzymes that catalyze this modification and show that arginine methylation inhibits the function of MBD2. Arginine methylation of MBD2 reduces MBD2-methyl-DNA complex formation, reduces MBD2-HDAC repression complex formation, and impairs the transcription repression function of MBD2 in cells. Our report provides a molecular description of a potential regulatory mechanism for an MBD protein family member. It is the first to demonstrate that protein arginine methyltransferases participate in the DNA methylation system of chromatin control.

Expression of oncomodulin does not lead to the transformation or immortalization of mammalian cells in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 517-525
Author(s):  
A.M. Mes-Masson ◽  
S. Masson ◽  
D. Banville ◽  
L. Chalifour
Keyword(s):  
Mammalian Cells ◽  
Mouse Kidney ◽  
Kidney Cells ◽  
T Antigens ◽  
Primary Mouse ◽  
Specific Antisera ◽  
Growth Properties ◽  
Metallothionein Promoter ◽  
Specific Mrna

A recombinant plasmid (pMTONCO) containing the coding sequences for rat oncomodulin under the direction of the metallothionein promoter was constructed. pMTONCO was co-transfected with the pSV2-NEO plasmid into primary mouse kidney cells or Rat-1 cells using the calcium phosphate technique and stable transformants were isolated after selection with G418. Transcription from the metallothionein promoter was inducible with heavy metals and produced an oncomodulin-specific mRNA. The presence of oncomodulin protein in stable cell lines was verified by immunoprecipitation with specific antisera. While a plasmid encoding the polyomavirus T-antigens was able to prolong the life-span of primary mouse kidney cells in culture, no equivalent activity was noted when the pMTONCO plasmid was used to transfect primary cells. When expressed in Rat-1 cells, oncomodulin did not affect the growth properties of these cells, nor did it predispose cells to higher frequencies of oncogenic transformation to a viral oncogene. We conclude that oncomodulin is neither an immortalizing nor transforming agent in vitro.

scholarly journals The coupling of translational control and stress responses

2020 ◽  
Vol 168 (2) ◽  
pp. 93-102 ◽  
Author(s):  
Ryan Houston ◽  
Shiori Sekine ◽  
Yusuke Sekine
Keyword(s):  
Stress Response ◽  
Stress Responses ◽  
Translational Control ◽  
Messenger Rna ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Translation Rate ◽  
Nucleolar Stress ◽  
Multistep Process ◽  
Aminoacyl Transfer

Abstract The translation of messenger RNA (mRNA) into protein is a multistep process by which genetic information transcribed into an mRNA is decoded to produce a specific polypeptide chain of amino acids. Ribosomes play a central role in translation by coordinately working with various translation regulatory factors and aminoacyl-transfer RNAs. Various stresses attenuate the ribosomal synthesis in the nucleolus as well as the translation rate in the cytosol. To efficiently reallocate cellular energy and resources, mammalian cells are endowed with mechanisms that directly link the suppression of translation-related processes to the activation of stress adaptation programmes. This review focuses on the integrated stress response (ISR) and the nucleolar stress response (NSR) both of which are activated by various stressors and selectively upregulate stress-responsive transcription factors. Emerging findings have delineated the detailed molecular mechanisms of the ISR and NSR and expanded their physiological and pathological significances.

scholarly journals Cytoskeletal proteins in the cell nucleus: a special nuclear actin perspective

2019 ◽  
Vol 30 (15) ◽  
pp. 1781-1785 ◽  
Author(s):  
Piergiorgio Percipalle ◽  
Maria Vartiainen
Keyword(s):  
Gene Expression ◽  
Cell Nucleus ◽  
Cell Biology ◽  
Genome Stability ◽  
Actin Polymerization ◽  
Nuclear Architecture ◽  
Nuclear Lamina ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Special Focus

The emerging role of cytoskeletal proteins in the cell nucleus has become a new frontier in cell biology. Actin and actin-binding proteins regulate chromatin and gene expression, but importantly they are beginning to be essential players in genome organization. These actin-based functions contribute to genome stability and integrity while affecting DNA replication and global transcription patterns. This is likely to occur through interactions of actin with nuclear components including nuclear lamina and subnuclear organelles. An exciting future challenge is to understand how these actin-based genome-wide mechanisms may regulate development and differentiation by interfering with the mechanical properties of the cell nucleus and how regulated actin polymerization plays a role in maintaining nuclear architecture. With a special focus on actin, here we summarize how cytoskeletal proteins operate in the nucleus and how they may be important to consolidate nuclear architecture for sustained gene expression or silencing.

Sign in / Sign up

Export Citation Format

Share Document