Rnai Pathway Participates Into Chromosome Segregation In

Total Page:16

File Type:pdf, Size:1020Kb

Rnai Pathway Participates Into Chromosome Segregation In OPEN Citation: Cell Discovery (2015) 1, 15029; doi:10.1038/celldisc.2015.29 ARTICLE www.nature.com/celldisc RNAi pathway participates in chromosome segregation in mammalian cells Chuan Huang, Xiaolin Wang, Xu Liu, Shuhuan Cao, Ge Shan School of Life Sciences, CAS Key Laboratory of Brain Function and Disease, University of Science and Technology of China, Hefei, China The RNAi machinery is a mighty regulator in a myriad of life events. Despite lines of evidence that small RNAs and components of the RNAi pathway may be associated with structure and behavior of mitotic chromosomes in diverse organisms, a direct role of the RNAi pathway in mammalian mitotic chromosome segregation remains elusive. Here we report that Dicer and AGO2, two central components of the mammalian RNAi pathway, participate in the chromosome segregation. Knockdown of Dicer or AGO2 results in a higher incidence of chromosome lagging, and this effect is independent from microRNAs as examined with DGCR8 knockout cells. Further investigation has revealed that α-satellite RNA, a noncoding RNA derived from centromeric repeat region, is managed by AGO2 under the guidance of endogenous small interference RNAs (ASAT siRNAs) generated by Dicer. Furthermore, the slicer activity of AGO2 is essential for the chromosome segregation. Level and distribution of chromosome-associated α-satellite RNA have crucial regulatory effect on the localization of centromeric proteins such as centromere protein C1 (CENPC1). With these results, we also provide a paradigm in which the RNAi pathway participates in vital cellular events through the maintenance of level and distribution of noncoding RNAs in cells. Keywords: RNAi pathway; chromosome segregation; CENPC1; AGO2; Dicer; α-satellite RNA; ASAT siRNA Cell Discovery (2015) 1, 15029; doi:10.1038/celldisc.2015.29; published online 20 October 2015 Introduction The core centromere of mammalian chromosome is the region for the assembly of kinetochore, and it The centromere is a special region of chromosome consists of α-satellite repeats in human and minor needed for successful assembly of kinetochore and satellite repeats in mice. Centromere protein B chromosome attachment to spindle [1–4]. Centromeres (CENP-B) is a highly conserved centromeric DNA- consist of long stretch of species-specific DNA repeats binding protein recognizing and binding to a 17-bp in most eukaryotes, and it has been known for more sequence (CENP-B box) in the centromeric α-satellite than a decade that some transcripts are derived from DNA [5, 6]. In vertebrates and mammals, studies have centromeric repeat regions in multiple eukaryotic demonstrated that transcripts from the centromeric organisms investigated [2, 3]. Although the organiza- core associate with centromeric core proteins to serve tion of centromere DNA as repeats is largely conserved as integral components of the centromere and in eukaryotes, the repeat sequences, transcripts from kinetochore [2, 3, 7–10]. Several studies have shown the repeats and the molecular mechanisms underneath that RNA polymerase II-dependent α-satellite RNA is the centromere/kinetochore assembly are not that an integral part of centromere and exerts an important conserved among different species. function in the kinetochore assembly [7–9]. Centromeric α-satellite RNA associates directly with centromere protein C1 (CENPC1) and facilitates nucleoprotein assembly at the mitotic centromere Correspondence: Ge Shan [7, 11–13]. CENPC1 has both RNA-binding and Tel: +86-551-63606274; Fax: +86-551-63606274; E-mail: [email protected] DNA-binding capacity [7, 8]. This protein is essential Received 5 March 2015; accepted 10 September 2015 for the integrity and localization of kinetochore, and RNAi pathway participates in chromosome segregation 2 loss of CENPC1 induces chromosome lagging [14, 15]. Results In mice, centromeric noncoding RNA (ncRNAs) of 120 nucleotides (nt) associate with centromeres Dicer or AGO2 knockdown leads to defect in and accumulate under stress or differentiation condi- mammalian chromosome segregation tions [16]. Components of kinetochore are recruited to We started to investigate roles of the RNAi pathway minor satellite repeat RNA by CENP-A [17, 18]. in chromosome segregation by knocking down Dicer In mouse embryonic stem cells, loss of Dicer reduces or AGO2 with siRNAs. Dicer or AGO2 knockdown the level of small double-stranded RNA from cen- resulted in chromosome lagging in mitotic human tromeric repeats [19]. In a chicken–human hybrid cell RPE-1 and HeLa cells (Figure 1a and Supplementary line, loss of Dicer results in a higher level of transcripts Figure S1A–E). As a comparison and also a positive from α-satellite sequences [20]. control, knocking down of CENPC1 resulted in higher The RNA interference (RNAi)/microRNA (miRNA) incidence of chromosome lagging (Figure 1a and pathway in mammalian cells is mediated by a cascade of Supplementary Figure S1B), and this phenomenon was enzymatic reactions [21–23]. First, the large primary consistent with early studies about roles of CENPC1 miRNA transcripts are processed by a complex of [14, 15, 26]. Furthermore, the mRNA and protein DGCR8 and Drosha into miRNA precursors of ~ 70 nt levels of CENPC1 were not significantly changed when with a hairpin structure in the nucleus. miRNA pre- knocking down the Dicer or AGO2, respectively cursors are then transported into the cytoplasm and get (Figure 1b). cut by Dicer to give rise to mature miRNAs. Endo- Functions of Dicer and AGO2 are largely associated genous or exogenous double-stranded RNA or short with two classes of small RNA, miRNA and siRNA hairpin RNA can also be processed by Dicer to form [23]. To see whether miRNAs are important, we next small interference RNA (siRNA). Both miRNA and carried out AGO2 and Dicer knockdown experiments siRNA can be loaded into RNA-induced silencing in DGCR8 knockout MEF cells [27–30]. It is well complex (RISC) with its core component as AGO2. known that these cells lack functional miRNAs, as Inside the siRNA-programmed RISC, the siRNA trig- DGCR8 is a critical component of microprocessor for gers the degradation of targeted complementary RNA the production of pre-miRNA from pri-miRNA with the slicer activity of AGO2. Whereas in the [27–30] (Supplementary Figure S1F). In DGCR8 miRISC (miRNA-programmed RISC), miRNA generally knockout cells, AGO2 or Dicer knockdown caused a suppresses the translation or promotes the degradation higher incidence of chromosome lagging (Figure 1c of the target mRNA by incomplete complementation. and Supplementary Figure S1G). Previous results showed that small RNAs and Interestingly, knocking down the other Argonaute components of the RNAi pathway might be associated family members including AGO1, AGO3 and AGO4 with mitotic chromosome segregation [1–3, 24, 25], did not induce chromosome lagging (Supplementary although the mechanism of how the RNAi machinery Figure S2). It seems that Dicer, as well as AGO2 rather participates into this event remains elusive. In this than the other Argonautes, possesses unique function study, we set out to investigate whether the RNAi in mitotic chromosome segregation. pathway participates in mitotic chromosome Taken together, our results demonstrated that segregation directly. We found that knockdown of AGO2 and Dicer acted in mammalian mitotic chro- Dicer or AGO2 resulted in chromosome lagging. With mosome segregation. Their roles in this cellular event a DGCR8 knockout cell line, we demonstrated that may be independent from miRNAs. this effect was largely independent from miRNAs. The RNAi pathway under the guidance of endogenous AGO2 but not dicer is a chromosomal protein siRNAs kept the level of α-satellite RNA balanced for To investigate further roles of Dicer and AGO2 on precise centromere/kinetochore formation. In addition, mitotic chromosome segregation, we isolated human the slicer activity of AGO2 was essential, indicating mitotic metaphase chromosomes with gradient further that a siRNA rather than a miRNA pathway centrifugation (Supplementary Figure S3A) [31]. was involved. Our data suggest that AGO2 guided by Results from flow cytometry and immunostaining of endogenous siRNAs generated by Dicer controls chromosome marker (Phos-histone H3) indicated that the level and localization of chromosomal α-satellite the isolated chromosomes were of high purity RNA to direct the deposition of important centromeric (Supplementary Figure S3B and C). Chromosomes factors such as CENPC1 for mitotic chromosome isolated with gradient centrifugation were mostly used segregation in mammalian cells. for western blot analyses and RNA isolation in this Cell Discovery | www.nature.com/celldisc Chuan Huang et al. 3 research. Western blot analysis with chromosome AGO2 or Dicer antibodies on mitotic chromosomes. samples showed that AGO2 rather than Dicer was a Metaphase chromosome spreads were prepared by chromosomal protein (Figure 1d). For further con- dropping for immunostaining assay. This method was firmation, we applied an immunostaining assay with used for all chromosomal immunofluorescent (IF) 120% *** ** ** ** ** RPE-1 cell 100% Scramble siRNA AGO2 siRNA-1 DICER siRNA-1 CENPC1 siRNA-1 80% 60% Normal 40% Chromosome AGO2 siRNA-2 DICER siRNA-2 CENPC1 siRNA-2 20% lagging Percentage of cells 0% AGO2 siRNA-1 AGO2 siRNA-2 Scramble siRNADICER siRNA-1 DICER siRNA-2 CENPC1 siRNA-1CENPC1 siRNA-2 qPCR of CENPC1 mRNA Western blot 1.2 1 scrambleAGO2 siRNA siRNADICER siRNACENPC1 siRNA 0.8 Scramble siRNA CENPC1 0.6 AGO2 siRNA DICER siRNA 1.00~0.80 ~0.85 ~0.14 0.4 ** CENPC1 siRNA Relative RNA level GAPDH 0.2 (Normalized with GAPDH) 0 120% *** ** 100% 80% MEF (DGCR8-null) cell Scramble siRNA AGO2 siRNA DICER siRNA CENPC1 siRNA 60% Normal 40% Chromosome Percentage of cells lagging 20% 0% AGO2 siRNADICER siRNA Scramble siRNA CENPC1 siRNA WCL CYTO CHR NUC 1/6 1/6 DAPI AGO2 Merge AGO2 DICER DAPI DICER Merge Phos-Histone H3 HADC2 ACTB Cell Discovery | www.nature.com/celldisc RNAi pathway participates in chromosome segregation 4 staining and RNA fluorescence in situ hybridization lengths in an array of human cells [33, 34], and our (FISH) in this study, unless specified.
Recommended publications
  • Epigenetic Control of Mammalian Centromere Protein Binding: Does DNA Methylation Have a Role?
    Journal of Cell Science 109, 2199-2206 (1996) 2199 Printed in Great Britain © The Company of Biologists Limited 1996 JCS3386 Epigenetic control of mammalian centromere protein binding: does DNA methylation have a role? Arthur R. Mitchell*, Peter Jeppesen, Linda Nicol†, Harris Morrison and David Kipling MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK *Author for correspondence (internet [email protected]) †Present address: MRC Reproductive Biology Unit, Edinburgh, UK SUMMARY Chromosome 1 of the inbred mouse strain DBA/2 has a block of minor satellite DNA sequences on chromosome 1. polymorphism associated with the minor satellite DNA at The binding of the CENP-E protein does not appear to be its centromere. The more terminal block of satellite DNA affected by demethylation of the minor satellite sequences. sequences on this chromosome acts as the centromere as We present a model to explain these observations. This shown by the binding of CREST ACA serum, anti-CENP- model may also indicate the mechanism by which the B and anti-CENP-E polyclonal sera. Demethylation of the CENP-B protein recognises specific sites within the arrays minor satellite DNA sequences accomplished by growing of minor satellite DNA on mouse chromosomes. cells in the presence of the drug 5-aza-2′-deoxycytidine results in a redistribution of the CENP-B protein. This protein now binds to an enlarged area on the more terminal Key words: Centromere satellite DNA, Demethylation, Centromere block and in addition it now binds to the more internal antibody INTRODUCTION A common feature of many mammalian pericentromeric domains is that they contain families of repetitive DNA The centromere of mammalian chromosomes is recognised at sequences (Singer, 1982).
    [Show full text]
  • Gene Knockdown of CENPA Reduces Sphere Forming Ability and Stemness of Glioblastoma Initiating Cells
    Neuroepigenetics 7 (2016) 6–18 Contents lists available at ScienceDirect Neuroepigenetics journal homepage: www.elsevier.com/locate/nepig Gene knockdown of CENPA reduces sphere forming ability and stemness of glioblastoma initiating cells Jinan Behnan a,1, Zanina Grieg b,c,1, Mrinal Joel b,c, Ingunn Ramsness c, Biljana Stangeland a,b,⁎ a Department of Molecular Medicine, Institute of Basic Medical Sciences, The Medical Faculty, University of Oslo, Oslo, Norway b Norwegian Center for Stem Cell Research, Department of Immunology and Transfusion Medicine, Oslo University Hospital, Oslo, Norway c Vilhelm Magnus Laboratory for Neurosurgical Research, Institute for Surgical Research and Department of Neurosurgery, Oslo University Hospital, Oslo, Norway article info abstract Article history: CENPA is a centromere-associated variant of histone H3 implicated in numerous malignancies. However, the Received 20 May 2016 role of this protein in glioblastoma (GBM) has not been demonstrated. GBM is one of the most aggressive Received in revised form 23 July 2016 human cancers. GBM initiating cells (GICs), contained within these tumors are deemed to convey Accepted 2 August 2016 characteristics such as invasiveness and resistance to therapy. Therefore, there is a strong rationale for targeting these cells. We investigated the expression of CENPA and other centromeric proteins (CENPs) in Keywords: fi CENPA GICs, GBM and variety of other cell types and tissues. Bioinformatics analysis identi ed the gene signature: fi Centromeric proteins high_CENP(AEFNM)/low_CENP(BCTQ) whose expression correlated with signi cantly worse GBM patient Glioblastoma survival. GBM Knockdown of CENPA reduced sphere forming ability, proliferation and cell viability of GICs. We also Brain tumor detected significant reduction in the expression of stemness marker SOX2 and the proliferation marker Glioblastoma initiating cells and therapeutic Ki67.
    [Show full text]
  • Centromere RNA Is a Key Component for the Assembly of Nucleoproteins at the Nucleolus and Centromere
    Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere Lee H. Wong,1,3 Kate H. Brettingham-Moore,1 Lyn Chan,1 Julie M. Quach,1 Melisssa A. Anderson,1 Emma L. Northrop,1 Ross Hannan,2 Richard Saffery,1 Margaret L. Shaw,1 Evan Williams,1 and K.H. Andy Choo1 1Chromosome and Chromatin Research Laboratory, Murdoch Childrens Research Institute & Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Parkville 3052, Victoria, Australia; 2Peter MacCallum Research Institute, St. Andrew’s Place, East Melbourne, Victoria 3002, Australia The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. Here, we demonstrate that centromeric ␣-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I–dependent manner. The nucleolar targeting of CENPC1 and INCENP requires ␣-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following ␣-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, we have identified the nucleolar localization sequences on CENPC1 and INCENP. We present evidence that CENPC1 is an RNA-associating protein that binds ␣-satellite RNA by an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and “RNA replenishment” experiments, we show that ␣-satellite RNA is a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere.
    [Show full text]
  • PDF Output of CLIC (Clustering by Inferred Co-Expression)
    PDF Output of CLIC (clustering by inferred co-expression) Dataset: Num of genes in input gene set: 13 Total number of genes: 16493 CLIC PDF output has three sections: 1) Overview of Co-Expression Modules (CEMs) Heatmap shows pairwise correlations between all genes in the input query gene set. Red lines shows the partition of input genes into CEMs, ordered by CEM strength. Each row shows one gene, and the brightness of squares indicates its correlations with other genes. Gene symbols are shown at left side and on the top of the heatmap. 2) Details of each CEM and its expansion CEM+ Top panel shows the posterior selection probability (dataset weights) for top GEO series datasets. Bottom panel shows the CEM genes (blue rows) as well as expanded CEM+ genes (green rows). Each column is one GEO series dataset, sorted by their posterior probability of being selected. The brightness of squares indicates the gene's correlations with CEM genes in the corresponding dataset. CEM+ includes genes that co-express with CEM genes in high-weight datasets, measured by LLR score. 3) Details of each GEO series dataset and its expression profile: Top panel shows the detailed information (e.g. title, summary) for the GEO series dataset. Bottom panel shows the background distribution and the expression profile for CEM genes in this dataset. Overview of Co-Expression Modules (CEMs) with Dataset Weighting Scale of average Pearson correlations Num of Genes in Query Geneset: 13. Num of CEMs: 1. 0.0 0.2 0.4 0.6 0.8 1.0 Cenpk Cenph Cenpp Cenpu Cenpn Cenpq Cenpl Apitd1
    [Show full text]
  • Cellular and Molecular Signatures in the Disease Tissue of Early
    Cellular and Molecular Signatures in the Disease Tissue of Early Rheumatoid Arthritis Stratify Clinical Response to csDMARD-Therapy and Predict Radiographic Progression Frances Humby1,* Myles Lewis1,* Nandhini Ramamoorthi2, Jason Hackney3, Michael Barnes1, Michele Bombardieri1, Francesca Setiadi2, Stephen Kelly1, Fabiola Bene1, Maria di Cicco1, Sudeh Riahi1, Vidalba Rocher-Ros1, Nora Ng1, Ilias Lazorou1, Rebecca E. Hands1, Desiree van der Heijde4, Robert Landewé5, Annette van der Helm-van Mil4, Alberto Cauli6, Iain B. McInnes7, Christopher D. Buckley8, Ernest Choy9, Peter Taylor10, Michael J. Townsend2 & Costantino Pitzalis1 1Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Departments of 2Biomarker Discovery OMNI, 3Bioinformatics and Computational Biology, Genentech Research and Early Development, South San Francisco, California 94080 USA 4Department of Rheumatology, Leiden University Medical Center, The Netherlands 5Department of Clinical Immunology & Rheumatology, Amsterdam Rheumatology & Immunology Center, Amsterdam, The Netherlands 6Rheumatology Unit, Department of Medical Sciences, Policlinico of the University of Cagliari, Cagliari, Italy 7Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK 8Rheumatology Research Group, Institute of Inflammation and Ageing (IIA), University of Birmingham, Birmingham B15 2WB, UK 9Institute of
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • The Genetic Program of Pancreatic Beta-Cell Replication in Vivo
    Page 1 of 65 Diabetes The genetic program of pancreatic beta-cell replication in vivo Agnes Klochendler1, Inbal Caspi2, Noa Corem1, Maya Moran3, Oriel Friedlich1, Sharona Elgavish4, Yuval Nevo4, Aharon Helman1, Benjamin Glaser5, Amir Eden3, Shalev Itzkovitz2, Yuval Dor1,* 1Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel 2Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. 3Department of Cell and Developmental Biology, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 4Info-CORE, Bioinformatics Unit of the I-CORE Computation Center, The Hebrew University and Hadassah, The Institute for Medical Research Israel- Canada, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel 5Endocrinology and Metabolism Service, Department of Internal Medicine, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel *Correspondence: [email protected] Running title: The genetic program of pancreatic β-cell replication 1 Diabetes Publish Ahead of Print, published online March 18, 2016 Diabetes Page 2 of 65 Abstract The molecular program underlying infrequent replication of pancreatic beta- cells remains largely inaccessible. Using transgenic mice expressing GFP in cycling cells we sorted live, replicating beta-cells and determined their transcriptome. Replicating beta-cells upregulate hundreds of proliferation- related genes, along with many novel putative cell cycle components. Strikingly, genes involved in beta-cell functions, namely glucose sensing and insulin secretion were repressed. Further studies using single molecule RNA in situ hybridization revealed that in fact, replicating beta-cells double the amount of RNA for most genes, but this upregulation excludes genes involved in beta-cell function.
    [Show full text]
  • Satellite DNA at the Centromere Is Dispensable for Segregation Fidelity
    G C A T T A C G G C A T genes Brief Report Satellite DNA at the Centromere Is Dispensable for Segregation Fidelity Annalisa Roberti, Mirella Bensi, Alice Mazzagatti, Francesca M. Piras, Solomon G. Nergadze , Elena Giulotto * and Elena Raimondi * Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Via Ferrata 1, 27100 Pavia, Italy; [email protected] (A.R.); [email protected] (M.B.); [email protected] (A.M.); [email protected] (F.M.P.); [email protected] (S.G.N.) * Correspondence: [email protected] (E.G.); [email protected] (E.R.) Received: 7 June 2019; Accepted: 19 June 2019; Published: 20 June 2019 Abstract: The typical vertebrate centromeres contain long stretches of highly repeated DNA sequences (satellite DNA). We previously demonstrated that the karyotypes of the species belonging to the genus Equus are characterized by the presence of satellite-free and satellite-based centromeres and represent a unique biological model for the study of centromere organization and behavior. Using horse primary fibroblasts cultured in vitro, we compared the segregation fidelity of chromosome 11, whose centromere is satellite-free, with that of chromosome 13, which has similar size and a centromere containing long stretches of satellite DNA. The mitotic stability of the two chromosomes was compared under normal conditions and under mitotic stress induced by the spindle inhibitor, nocodazole. Two independent molecular-cytogenetic approaches were used—the interphase aneuploidy analysis and the cytokinesis-block micronucleus assay. Both assays were coupled to fluorescence in situ hybridization with chromosome specific probes in order to identify chromosome 11 and chromosome 13, respectively.
    [Show full text]
  • Genome-Wide Screening Identifies Genes and Biological Processes
    Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 10-12-2018 Genome-Wide Screening Identifies Genes and Biological Processes Implicated in Chemoresistance and Oncogene-Induced Apoptosis Tengyu Ko Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Cancer Biology Commons, Cell Biology Commons, and the Genomics Commons Recommended Citation Ko, Tengyu, "Genome-Wide Screening Identifies Genes and Biological Processes Implicated in Chemoresistance and Oncogene- Induced Apoptosis" (2018). LSU Doctoral Dissertations. 4715. https://digitalcommons.lsu.edu/gradschool_dissertations/4715 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. GENOME-WIDE SCREENING IDENTIFIES GENES AND BIOLOGICAL PROCESSES IMPLICATED IN CHEMORESISTANCE AND ONCOGENE- INDUCED APOPTOSIS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical and Veterinary Medical Sciences through the Department of Comparative Biomedical Sciences by Tengyu Ko B.S., University of California, Santa Barbara 2010 December 2018 ACKNOWLEDGEMENTS I would like to express my sincerest gratitude to my major supervisor Dr. Shisheng Li for giving me the opportunity to join his team and the freedom to pursue projects. I appreciate all of his thoughts and efforts. Truly, none of these findings would be possible without his supervisions, supports, insightful discussions, and patience.
    [Show full text]
  • Identification and Characterization of Satellite Dnas in Two-Toed Sloths Of
    www.nature.com/scientificreports OPEN Identifcation and characterization of satellite DNAs in two‑toed sloths of the genus Choloepus (Megalonychidae, Xenarthra) Radarane Santos Sena1, Pedro Heringer1, Mirela Pelizaro Valeri1, Valéria Socorro Pereira2, Gustavo C. S. Kuhn1 & Marta Svartman1* Choloepus, the only extant genus of the Megalonychidae family, is composed of two living species of two‑toed sloths: Choloepus didactylus and C. hofmanni. In this work, we identifed and characterized the main satellite DNAs (satDNAs) in the sequenced genomes of these two species. SATCHO1, the most abundant satDNA in both species, is composed of 117 bp tandem repeat sequences. The second most abundant satDNA, SATCHO2, is composed of ~ 2292 bp tandem repeats. Fluorescence in situ hybridization in C. hofmanni revealed that both satDNAs are located in the centromeric regions of all chromosomes, except the X. In fact, these satDNAs present some centromeric characteristics in their sequences, such as dyad symmetries predicted to form secondary structures. PCR experiments indicated the presence of SATCHO1 sequences in two other Xenarthra species: the tree‑toed sloth Bradypus variegatus and the anteater Myrmecophaga tridactyla. Nevertheless, SATCHO1 is present as large tandem arrays only in Choloepus species, thus likely representing a satDNA exclusively in this genus. Our results reveal interesting features of the satDNA landscape in Choloepus species with the potential to aid future phylogenetic studies in Xenarthra and mammalian genomes in general. A signifcant part of eukaryotic genomes, ~ 30% in some plants to more than 50% in some insects and mammals, is composed of tandemly organized highly repetitive sequences, known as satellite DNAs (satDNAs) (reviewed in Ref.1).
    [Show full text]
  • 1 SUPPLEMENTAL DATA Figure S1. Poly I:C Induces IFN-Β Expression
    SUPPLEMENTAL DATA Figure S1. Poly I:C induces IFN-β expression and signaling. Fibroblasts were incubated in media with or without Poly I:C for 24 h. RNA was isolated and processed for microarray analysis. Genes showing >2-fold up- or down-regulation compared to control fibroblasts were analyzed using Ingenuity Pathway Analysis Software (Red color, up-regulation; Green color, down-regulation). The transcripts with known gene identifiers (HUGO gene symbols) were entered into the Ingenuity Pathways Knowledge Base IPA 4.0. Each gene identifier mapped in the Ingenuity Pathways Knowledge Base was termed as a focus gene, which was overlaid into a global molecular network established from the information in the Ingenuity Pathways Knowledge Base. Each network contained a maximum of 35 focus genes. 1 Figure S2. The overlap of genes regulated by Poly I:C and by IFN. Bioinformatics analysis was conducted to generate a list of 2003 genes showing >2 fold up or down- regulation in fibroblasts treated with Poly I:C for 24 h. The overlap of this gene set with the 117 skin gene IFN Core Signature comprised of datasets of skin cells stimulated by IFN (Wong et al, 2012) was generated using Microsoft Excel. 2 Symbol Description polyIC 24h IFN 24h CXCL10 chemokine (C-X-C motif) ligand 10 129 7.14 CCL5 chemokine (C-C motif) ligand 5 118 1.12 CCL5 chemokine (C-C motif) ligand 5 115 1.01 OASL 2'-5'-oligoadenylate synthetase-like 83.3 9.52 CCL8 chemokine (C-C motif) ligand 8 78.5 3.25 IDO1 indoleamine 2,3-dioxygenase 1 76.3 3.5 IFI27 interferon, alpha-inducible
    [Show full text]
  • CENP-B Dynamics at Centromeres Is Regulated by a Sumoylation
    bioRxiv preprint doi: https://doi.org/10.1101/245597; this version posted January 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 CENP-B dynamics at centromeres is regulated by a SUMOylation/ubiquitination and 2 proteasomal-dependent degradation mechanism involving the SUMO-targeted ubiquitin E3 3 ligase RNF4 4 Jhony El Maalouf 1,, Pascale Texier 1,4, Indri Erliandri 1,4, Camille Cohen 1, Armelle Corpet 1, Frédéric 5 Catez 2, Chris Boutell 3, Patrick Lomonte 1,* 6 7 1. Univ Lyon, Université Claude Bernard Lyon 1, CNRS UMR 5310, INSERM U 1217, LabEx DEVweCAN, 8 Institut NeuroMyoGène (INMG), team Chromatin Assembly, Nuclear Domains, Virus. F-69100, Lyon, 9 France 10 2. Univ Lyon, Université Claude Bernard Lyon 1, CNRS UMR 5286, INSERM U 1052, Centre de 11 Recherche en Cancérologie de Lyon. F-69000, Lyon, France 12 3. MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, Scotland (UK) 13 4. Contributed equally to this work. 14 15 * Corresponding author: [email protected] 16 17 18 Running title: SUMO & ubiquitin-dependent CENP-B dynamics 19 20 Word count: 6743 21 22 23 24 The English of parts of this document has been checked by at least two professional editors, both native 25 speakers of English. For a certificate, please see: 26 27 http://www.textcheck.com/certificate/gh7EcX 28 29 30 1 bioRxiv preprint doi: https://doi.org/10.1101/245597; this version posted January 9, 2018.
    [Show full text]