DOCSLIB.ORG

The Cnot Complex Contributes to the Maintenance of Genome Stability

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Cnot Complex Contributes to the Maintenance of Genome Stability THE CNOT COMPLEX CONTRIBUTES TO THE MAINTENANCE OF GENOME STABILITY By NAFISEH CHALABI HAGKARIM A thesis presented to the College of Medical and Dental Sciences, the University of Birmingham, for the degree of Doctor of Philosophy Institute of Cancer & Genomic Sciences College of Medical and Dental Sciences University of Birmingham December 2019 1 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract The yeast CCR4-NOT (CNOT in mammals) complex is a large (1.0-MDa) and highly conserved multifunctional set of proteins. It is involved in many different aspects of mRNA metabolism, including repression and activation of mRNA initiation, control of mRNA elongation, and deadenylation-dependent mRNA turnover; it also has a role in ubiquitin-protein transferase activity and histone methylation. Some studies have suggested that the yeast complex may be involved in the recognition and repair of DNA damage. To investigate whether similar properties are attributable to the mammalian complex we have examined the effects of inactivation of the complex on various aspects of the DNA damage response. Inactivation was achieved by depletion of CNOT1, the largest of the CNOT proteins, which forms a scaffold to the complex. Ablation of CNOT1 expression disrupts cell cycle progression through S and G2/M phases, which subsequently arrests the cell cycle in G1, with markedly elevated levels of cyclin E, p27 and p21. At later times, the cells appear to senesce and /or undergo autophagy. As expected, depletion of CNOT1 affects global transcription and can lead to transcription-dependent replication stress and R-loop formation. CNOT1 depletion can also affect DNA replication by reducing dNTP synthesis. Activity of the RNase H2 complex decreases following loss of CNOT1, which increases the sensitivity of genomic DNA to alkaline lysis due to an increase in embedded ribonucleotides. In addition, depletion of CNOT1 results in DNA damage as seen by comet assay, and formation of micronuclei. This is accompanied by activation of Chk2 in the absence of extraneous DNA damage. In this study, we have demonstrated that the CNOT complex contributes to the maintenance of genome stability and to the response to DNA damage. 2 To my parents 3 Acknowledgments I would like to thank many people for their help and support during my PhD, without them I might have given up a long time ago. I would like to begin with my wonderful supervisor Dr Roger Grand, who never stopped believing in me when the other people did! His encouraging support allowed me to grow as a research scientist. His advice on both research as well as on my career have been invaluable. He has also been a great friend to me, especially during the tough time in my PhD. I have experienced a very peaceful time with him. I cannot be more thankful. Many thanks to Grant Stewart for his help and encouragement. I cannot think of becoming a doctor without my best friend, my other half, my husband Ali. Frankly, I cannot express in words my massive appreciation of him who supported me in the worst and the best moments of my PhD. I took full advantage of his previous experience of doing a PhD by constant questioning (sometimes very stupid ones) and demanding requests; however, his never-ending patience and encouragement made me feel that I am still welcome to ask more. I would also like to acknowledge my parents to whom my life itself! I am not sure I would have made my life through without you. You were always there for me without hesitation and I apologise if I was not there for you when you needed me. My brother also deserves a line in here for his great help with my RNA-Seq bioinformatics data analysis, childhood partners in crime are now partners in science! My special thanks goes to Grant Stewart’ group members Martin, John, Rob, Sati and Danxu, Tanja’s group Angelo, Phil, Edward and Nick; Liza from Eva’s group, Marco and also Panagiotis whom were always on hand to offer scientific advice and to lend reagents and materials. “No gain no pain” this is what I have learned during my PhD, hats off to all doctors out there! 4 PUBLICATIONS AND CONFERENCES Chalabi Hagkarim, N., Ryan, E.L., Byrd, P.J., Hollingworth, R., Shimwell, N.J., Agathanggelou, A., Vavasseur, M., Kolbe, V., Speiseder, T., Dobner, T. and Stewart, G.S., (2018). Degradation of a novel DNA damage response protein, tankyrase 1 binding protein 1, following adenovirus infection. Journal of virology, 92 (12), pp. e02034-17. Chalabi Hagkarim, N., Eskandarian S., Stewart, G., Grand, R.J (2018). The Role of the Human CNOT1 Protein in the DNA Damage Response. Poster session presented at the BCGB symposium 2018. Birmingham-UK. Chalabi Hagkarim, N., Eskandarian S., Stewart, G., Grand, R.J (2019). The CNOT complex contributes to the maintenance of genome stability. Poster session presented at the BCGB symposium 2018. Birmingham-UK. Gerrard, G., Chalabi Hagkarim, N., Szydlo, R., Alikian, M., Alonso-Dominguez, J.M., Grinfeld, J., Hedgley, C., O'Brien, S., Clark, R.E., Apperley, J., Foroni, L. et al., 2014, Transcript Levels of the Hedgehog Pathway Members PTCH1 and SMO are predictive of Imatinib Failure in pre-treatment Chronic Myeloid Leukaemia, 19th Congress of the European-Hematology-Association, Publisher: Ferrata Storti Foundation , pp. 510-510, ISSN: 0390-6078. Chalabi Hagkarim, N., Eskandarian S., Stewart, G., Grand, R.J (2017). The Role of the Human CNOT1 Protein in the DNA Damage Response. Poster session presented at the 1st Cancer symposium Bath. Bath-UK. Chalabi Hagkarim, N., Ryan, E.L Stewart, G., Grand, R.J (2017). Degradation of a novel DNA damage response protein, tankyrase 1 binding protein 1 (Tab182), following adenovirus infection. Poster session presented at the DNA Tumour Virus 2017. Birmingham-UK. Chalabi Hagkarim, N., Eskandarian S., Stewart, G., Grand, R.J (2017). The Role of the Human CNOT1 Protein in the DNA Damage Response. Oral presentation at the Midlands Cell Cycle & Cytoskeleton workshop 2017. Nottingham-UK. Chalabi Hagkarim, N., Eskandarian S., Stewart, G., Grand, R.J (2017). The Role of the Human CNOT1 Protein in the DNA Damage Response. Poster session presented at EMBO DDR symposium 2017. Greece-Athene. 5 TABLE OF CONTENTS 1 INTRODUCTION ................................................................................................................. 20 1.1 The CCR4-NOT complex ............................................................................................ 20 1.1.1 CNOT1 .................................................................................................................... 26 1.1.2 Deadenylation of mRNAs and the CNOT complex ................................................ 29 1.1.3 Transcription initiation and the CNOT complex .................................................... 32 1.1.4 Transcription elongation and the CNOT complex ................................................. 35 1.2 Cell cycle .................................................................................................................... 38 1.2.1 The Cell Cycle Checkpoints .................................................................................... 41 1.3 DNA Replication ........................................................................................................ 51 1.3.1 DNA origin licencing and activation....................................................................... 51 1.3.2 DNA replication stress response ........................................................................... 55 1.3.3 Activation of the ATR/Chk1 pathway and fork stabilization ................................. 55 1.3.4 Replication forks reversal ...................................................................................... 58 1.3.5 Oncogene-induced replication stress .................................................................... 65 1.4 Transcription-induced stress response ..................................................................... 75 1.4.1 Repair of Intra-strand crosslinks (ICLs) and DNA bulky adducts ........................... 75 1.4.2 Imbalanced rNTP / dNTP pool ratio and DNA polymerase-mediated incorporation errors 80 1.4.3 Repair of mis-inserted rNMPs in genomic DNA by ribonucleotide excision repair (RER) 83 1.4.4 Repair of TOP1-dependent single-stranded DNA (ssDNA) nicks ........................... 84 1.4.5 Repair of R-Loop-dependent genome instability .................................................. 87 1.5 Double-strand break repair ....................................................................................... 90 1.5.1 Homologous recombination (HR) pathway ........................................................... 92 1.5.2 Non-Homologous End Joining ............................................................................... 95 1.5.3 Fanconi anaemia pathway and interstrand cross-links ......................................... 97 1.6 Hypothesis and aims ................................................................................................
Load more

Recommended publications
  • Analysis of Trans Esnps Infers Regulatory Network Architecture
    Analysis of trans eSNPs infers regulatory network architecture Anat Kreimer Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2014 © 2014 Anat Kreimer All rights reserved ABSTRACT Analysis of trans eSNPs infers regulatory network architecture Anat Kreimer eSNPs are genetic variants associated with transcript expression levels. The characteristics of such variants highlight their importance and present a unique opportunity for studying gene regulation. eSNPs affect most genes and their cell type specificity can shed light on different processes that are activated in each cell. They can identify functional variants by connecting SNPs that are implicated in disease to a molecular mechanism. Examining eSNPs that are associated with distal genes can provide insights regarding the inference of regulatory networks but also presents challenges due to the high statistical burden of multiple testing. Such association studies allow: simultaneous investigation of many gene expression phenotypes without assuming any prior knowledge and identification of unknown regulators of gene expression while uncovering directionality. This thesis will focus on such distal eSNPs to map regulatory interactions between different loci and expose the architecture of the regulatory network defined by such interactions. We develop novel computational approaches and apply them to genetics-genomics data in human. We go beyond pairwise interactions to define network motifs, including regulatory modules and bi-fan structures, showing them to be prevalent in real data and exposing distinct attributes of such arrangements. We project eSNP associations onto a protein-protein interaction network to expose topological properties of eSNPs and their targets and highlight different modes of distal regulation.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • CNOT4 Antibody (Pab)
    21.10.2014CNOT4 antibody (pAb) Rabbit Anti-Human/Mouse/Rat CCR4-NOT Transcription Complex Subunit 4 (NOT4, NOT4H) Instruction Manual Catalog Number PK-AB718-4813 Synonyms CNOT4 Antibody: CCR4-NOT transcription complex subunit 4, NOT4, NOT4H Description CNOT4 is a component of the CCR4-NOT transcription complex, a complex that is implicated in the repression of RNA polymerase II transcription. In the CCR4-NOT complex, CNOT4 acts as an E3 ubiquitin-protein ligase and interacts with a subset of E2 ubiquitin-conjugating enzymes through a unique C4C4 RING domain. This E3 ligase activity was shown to be dependent on the selective and specific interaction with the ubiquitin conjugating enzyme UbcH5B. In yeast, mutations in CNOT4 that prevented its interaction with the UbcH5B homolog UBC4 caused increased sensitivity to hydroxyurea, heat shock, and hygromycin B, suggesting that CNOT4 and UbcH5B are involved in stress response in vivo. Multiple isoforms of CNOT4 are known to exist. Quantity 100 µg Source / Host Rabbit Immunogen CNOT4 antibody was raised against a 19 amino acid peptide near the amino terminus of the human CNOT4. Purification Method Affinity chromatography purified via peptide column. Clone / IgG Subtype Polyclonal antibody Species Reactivity Human, Mouse, Rat Specificity Formulation Antibody is supplied in PBS containing 0.02% sodium azide. Reconstitution During shipment, small volumes of antibody will occasionally become entrapped in the seal of the product vial. For products with volumes of 200 μl or less, we recommend gently tapping the vial on a hard surface or briefly centrifuging the vial in a tabletop centrifuge to dislodge any liquid in the container’s cap.
    [Show full text]
  • Whole Exome Sequencing in Families at High Risk for Hodgkin Lymphoma: Identification of a Predisposing Mutation in the KDR Gene
    Hodgkin Lymphoma SUPPLEMENTARY APPENDIX Whole exome sequencing in families at high risk for Hodgkin lymphoma: identification of a predisposing mutation in the KDR gene Melissa Rotunno, 1 Mary L. McMaster, 1 Joseph Boland, 2 Sara Bass, 2 Xijun Zhang, 2 Laurie Burdett, 2 Belynda Hicks, 2 Sarangan Ravichandran, 3 Brian T. Luke, 3 Meredith Yeager, 2 Laura Fontaine, 4 Paula L. Hyland, 1 Alisa M. Goldstein, 1 NCI DCEG Cancer Sequencing Working Group, NCI DCEG Cancer Genomics Research Laboratory, Stephen J. Chanock, 5 Neil E. Caporaso, 1 Margaret A. Tucker, 6 and Lynn R. Goldin 1 1Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; 2Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; 3Ad - vanced Biomedical Computing Center, Leidos Biomedical Research Inc.; Frederick National Laboratory for Cancer Research, Frederick, MD; 4Westat, Inc., Rockville MD; 5Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; and 6Human Genetics Program, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD, USA ©2016 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2015.135475 Received: August 19, 2015. Accepted: January 7, 2016. Pre-published: June 13, 2016. Correspondence: [email protected] Supplemental Author Information: NCI DCEG Cancer Sequencing Working Group: Mark H. Greene, Allan Hildesheim, Nan Hu, Maria Theresa Landi, Jennifer Loud, Phuong Mai, Lisa Mirabello, Lindsay Morton, Dilys Parry, Anand Pathak, Douglas R. Stewart, Philip R. Taylor, Geoffrey S. Tobias, Xiaohong R. Yang, Guoqin Yu NCI DCEG Cancer Genomics Research Laboratory: Salma Chowdhury, Michael Cullen, Casey Dagnall, Herbert Higson, Amy A.
    [Show full text]
  • Dissertation
    Regulation of gene silencing: From microRNA biogenesis to post-translational modifications of TNRC6 complexes DISSERTATION zur Erlangung des DOKTORGRADES DER NATURWISSENSCHAFTEN (Dr. rer. nat.) der Fakultät Biologie und Vorklinische Medizin der Universität Regensburg vorgelegt von Johannes Danner aus Eggenfelden im Jahr 2017 Das Promotionsgesuch wurde eingereicht am: 12.09.2017 Die Arbeit wurde angeleitet von: Prof. Dr. Gunter Meister Johannes Danner Summary ‘From microRNA biogenesis to post-translational modifications of TNRC6 complexes’ summarizes the two main projects, beginning with the influence of specific RNA binding proteins on miRNA biogenesis processes. The fate of the mature miRNA is determined by the incorporation into Argonaute proteins followed by a complex formation with TNRC6 proteins as core molecules of gene silencing complexes. miRNAs are transcribed as stem-loop structured primary transcripts (pri-miRNA) by Pol II. The further nuclear processing is carried out by the microprocessor complex containing the RNase III enzyme Drosha, which cleaves the pri-miRNA to precursor-miRNA (pre-miRNA). After Exportin-5 mediated transport of the pre-miRNA to the cytoplasm, the RNase III enzyme Dicer cleaves off the terminal loop resulting in a 21-24 nt long double-stranded RNA. One of the strands is incorporated in the RNA-induced silencing complex (RISC), where it directly interacts with a member of the Argonaute protein family. The miRNA guides the mature RISC complex to partially complementary target sites on mRNAs leading to gene silencing. During this process TNRC6 proteins interact with Argonaute and recruit additional factors to mediate translational repression and target mRNA destabilization through deadenylation and decapping leading to mRNA decay.
    [Show full text]
  • Role and Regulation of the P53-Homolog P73 in the Transformation of Normal Human Fibroblasts
    Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Hofmann aus Aschaffenburg Würzburg 2007 Eingereicht am Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Dr. Martin J. Müller Gutachter: Prof. Dr. Michael P. Schön Gutachter : Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher, außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen Grade erworben und zu erwerben gesucht. Würzburg, Lars Hofmann Content SUMMARY ................................................................................................................ IV ZUSAMMENFASSUNG ............................................................................................. V 1. INTRODUCTION ................................................................................................. 1 1.1. Molecular basics of cancer .......................................................................................... 1 1.2. Early research on tumorigenesis ................................................................................. 3 1.3. Developing
    [Show full text]
  • Structural Model of the Ubch5b/CNOT4 Complex Revealed by Combining NMR, Mutagenesis, and Docking Approaches
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Structure, Vol. 12, 633–644, April, 2004, 2004 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.str.2004.03.004 Structural Model of the UbcH5B/CNOT4 Complex Revealed by Combining NMR, Mutagenesis, and Docking Approaches Cyril Dominguez,1 Alexandre M.J.J. Bonvin,1 Weissman, 2001). Different classes of E3 ligases have G. Sebastiaan Winkler,2 Frederik M.A. van Schaik,2 been found that mediate substrate ubiquitination. E3 H.Th. Marc Timmers,2 and Rolf Boelens1,* enzymes can be distinguished by their E2-interacting 1Department of NMR Spectroscopy domains, which include the HECT and the RING domains Bijvoet Center for Biomolecular Research (Glickman and Ciechanover, 2002; Pickart, 2001). There- Utrecht University fore, specific E2/E3 pairs are thought to be responsible Padualaan 8 for mediating target recognition and subsequent ubiqui- 3584 Utrect tination. Structures of nine E2 enzymes from different 2 Department of Physiological Chemistry species in the free form have been solved by X-ray University Medical Center Utrecht crystallography (Cook et al., 1992, 1993, 1997; Hamilton Universiteitsweg 100 et al., 2001; Jiang and Basavappa, 1999; Lin et al., 2002; 3584 CG Utrecht Tong et al., 1997; VanDemark et al., 2001; Worthylake The Netherlands et al., 1998) and one was solved by NMR (Miura et al., 2002). The structure of the human E2 enzyme UbcH5B, however, has not been determined. Furthermore, five Summary structures of E2 in complex with various other proteins have been solved by X-ray crystallography (Bernier-Vil- The protein CNOT4 possesses an N-terminal RING lamor et al., 2002; Huang et al., 1999; Moraes et al., finger domain that acts as an E3 ubiquitin ligase and 2001; VanDemark et al., 2001; Zheng et al., 2000).
    [Show full text]
  • Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity in Vivo Wi S
    Washington University School of Medicine Digital Commons@Becker Open Access Publications 2019 Importance of the conserved carboxyl-terminal CNOT1 binding domain to tristetraprolin activity in vivo Wi S. Lai Deborah J. Stumpo Melissa L. Wells Artion Gruzdev Stephanie N. Hicks See next page for additional authors Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Authors Wi S. Lai, Deborah J. Stumpo, Melissa L. Wells, Artion Gruzdev, Stephanie N. Hicks, Cindo O. Nicholson, Zhengfeng Yang, Roberta Faccio, Michael W. Webster, Lori A. Passmore, and Perry J. Blackshear RESEARCH ARTICLE crossm Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo Wi S. Lai,a Deborah J. Stumpo,a Melissa L. Wells,a Artiom Gruzdev,b Stephanie N. Hicks,a Cindo O. Nicholson,a Zhengfeng Yang,c,f* Roberta Faccio,c,f Michael W. Webster,d* Lori A. Passmore,d Perry J. Blacksheara,e Downloaded from aSignal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA bReproductive & Developmental Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA cDepartment of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, Missouri, USA dMRC Laboratory of Molecular Biology, Cambridge, United Kingdom eDepartments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina, USA fShriners Hospitals for Children, St. Louis, Missouri, USA ABSTRACT Tristetraprolin (TTP) is an anti-inflammatory protein that modulates the sta- http://mcb.asm.org/ bility of certain cytokine/chemokine mRNAs. After initial high-affinity binding to AU-rich elements in 3= untranslated regions of target mRNAs, mediated through its tandem zinc finger (TZF) domain, TTP promotes the deadenylation and ultimate decay of target tran- scripts.
    [Show full text]
  • Importance of CNOT8 Deadenylase Subunit in DNA Damage Responses Following Ionizing Radiation (IR)
    Reports of Biochemistry & Molecular Biology Vol.9, No.1, July 2020 Original article www.RBMB.net Importance of CNOT8 Deadenylase Subunit in DNA Damage Responses Following Ionizing Radiation (IR) Samira Eskandarian1,2, Roger Grand2, Shiva Irani*1, Mohsen Saeedi3, Reza Mirfakhraie4 Abstract Background: The Ccr4-Not protein complex (CNOT complex) is a key regulator of gene expression in eukaryotic cells. Ccr4-Not Complex is composed of at least nine conserved subunits in mammalian cells with two main enzymatic activities. CNOT8 is a subunit of the complex with deadenylase activity that interacts transiently with the CNOT6 or CNOT6L subunits. Here, we focused on the role of the human CNOT8 subunit in the DNA damage response (DDR). Methods: Cell viability was assessed to measure ATP level using a Cell Titer-Glo Luminescence reagent up to 4 days’ post CNOT8 siRNA transfection. In addition, expression level of phosphorylated proteins in signalling pathways were detected by western blotting and immunofluorescence microscopy. CNOT8- depleted Hela cells post- 3 Gy ionizing radiation (IR) treatment were considered as a control. Results: Our results from cell viability assays indicated a significant reduction at 72-hour post CNOT8 siRNA transfection (p= 0.04). Western blot analysis showed slightly alteration in the phosphorylation of DNA damage response (DDR) proteins in CNOT8-depleted HeLa cells following treatment with ionizing radiation (IR). Increased foci formation of H2AX, RPA, 53BP1, and RAD51 foci was observed after IR in CNOT8-depleted cells compared to the control cells. Conclusions: We conclude that CNOT8 deadenylase subunit is involved in the cellular response to DNA damage. Keywords: CNOT complex, CNOT8, DNA damage response, SiRNA.
    [Show full text]
  • Transcriptional Regulator CNOT3 Defines an Aggressive Colorectal Cancer Subtype
    Published OnlineFirst November 29, 2016; DOI: 10.1158/0008-5472.CAN-16-1346 Cancer Tumor and Stem Cell Biology Research Transcriptional Regulator CNOT3 Defines an Aggressive Colorectal Cancer Subtype Paloma Cejas1,2,3, Alessia Cavazza1,2, C.N. Yandava1, Victor Moreno3, David Horst4, Juan Moreno-Rubio3, Emilio Burgos5, Marta Mendiola3,5, Len Taing1, Ajay Goel6, Jaime Feliu3, and Ramesh A. Shivdasani1,2 Abstract Cancer cells exhibit dramatic alterations of chromatin organi- primarily to genes whose expression was affected by CNOT3 loss, zation at cis-regulatory elements, but the molecular basis, extent, and also at sites modulated in certain types of colorectal cancers. and impact of these alterations are still being unraveled. Here, we These target genes were implicated in ESC and cancer self-renewal identify extensive genome-wide modification of sites bearing the and fell into two distinct groups: those dependent on CNOT3 and active histone mark H3K4me2 in primary human colorectal MYC for optimal transcription and those repressed by CNOT3 cancers, as compared with corresponding benign precursor ade- binding and promoter hypermethylation. Silencing CNOT3 in nomas. Modification of certain colorectal cancer sites highlighted colorectal cancer cells resulted in replication arrest. In clinical þ the activity of the transcription factor CNOT3, which is known to specimens, early-stage tumors that included >5% CNOT3 cells control self-renewal of embryonic stem cells (ESC). In primary exhibited a correlation to worse clinical outcomes compared with colorectal cancer cells, we observed a scattered pattern of CNOT3 tumors with little to no CNOT3 expression. Together, our findings expression, as might be expected for a tumor-initiating cell mark- implicate CNOT3 in the coordination of colonic epithelial cell er.
    [Show full text]
  • NRF1) Coordinates Changes in the Transcriptional and Chromatin Landscape Affecting Development and Progression of Invasive Breast Cancer
    Florida International University FIU Digital Commons FIU Electronic Theses and Dissertations University Graduate School 11-7-2018 Decipher Mechanisms by which Nuclear Respiratory Factor One (NRF1) Coordinates Changes in the Transcriptional and Chromatin Landscape Affecting Development and Progression of Invasive Breast Cancer Jairo Ramos [email protected] Follow this and additional works at: https://digitalcommons.fiu.edu/etd Part of the Clinical Epidemiology Commons Recommended Citation Ramos, Jairo, "Decipher Mechanisms by which Nuclear Respiratory Factor One (NRF1) Coordinates Changes in the Transcriptional and Chromatin Landscape Affecting Development and Progression of Invasive Breast Cancer" (2018). FIU Electronic Theses and Dissertations. 3872. https://digitalcommons.fiu.edu/etd/3872 This work is brought to you for free and open access by the University Graduate School at FIU Digital Commons. It has been accepted for inclusion in FIU Electronic Theses and Dissertations by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. FLORIDA INTERNATIONAL UNIVERSITY Miami, Florida DECIPHER MECHANISMS BY WHICH NUCLEAR RESPIRATORY FACTOR ONE (NRF1) COORDINATES CHANGES IN THE TRANSCRIPTIONAL AND CHROMATIN LANDSCAPE AFFECTING DEVELOPMENT AND PROGRESSION OF INVASIVE BREAST CANCER A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in PUBLIC HEALTH by Jairo Ramos 2018 To: Dean Tomás R. Guilarte Robert Stempel College of Public Health and Social Work This dissertation, Written by Jairo Ramos, and entitled Decipher Mechanisms by Which Nuclear Respiratory Factor One (NRF1) Coordinates Changes in the Transcriptional and Chromatin Landscape Affecting Development and Progression of Invasive Breast Cancer, having been approved in respect to style and intellectual content, is referred to you for judgment.
    [Show full text]
  • Inhibition of Notch Signaling Attenuates Pituitary Adenoma Growth in Nude Mice
    26 1 Endocrine-Related Z-B Lautaro et al. Notch inhibition and pituitary 26:1 13–29 Cancer tumor growth RESEARCH Inhibition of Notch signaling attenuates pituitary adenoma growth in Nude mice Lautaro Zubeldía-Brenner1, Catalina De Winne1, Sofía Perrone2, Santiago A Rodríguez-Seguí3,4, Christophe Willems5, Ana María Ornstein1, Isabel Lacau-Mengido1, Hugo Vankelecom5, Carolina Cristina2* and Damasia Becu-Villalobos1* 1Instituto de Biología y Medicina Experimental, IBYME-CONICET, Buenos Aires, Argentina 2Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires, CITNOBA (UNNOBA-CONICET), Universidad Nacional del Noroeste de la Provincia de Buenos Aires, Buenos Aires, Argentina 3Departamento de Fisiología y Biología Molecular y Celular, Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina 4CONICET-Universidad de Buenos Aires, Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), Buenos Aires, Argentina 5Department of Development and Regeneration, Cluster Stem Cell and Developmental Biology, Unit of Stem Cell Research, KU Leuven (University of Leuven), Leuven, Belgium Correspondence should be addressed to D Becu-Villalobos: [email protected] *(C Cristina and D Becu-Villalobos contributed equally) Abstract Preclinical and clinical studies support that Notch signaling may play an important Key Words oncogenic role in cancer, but there is scarce information for pituitary tumors. f DAPT We therefore undertook a functional study to evaluate Notch participation in f pituitary pituitary adenoma growth. Tumors generated in Nude mice by subcutaneous GH3 f angiogenesis somatolactotrope cell injection were treated in vivo with DAPT, a γ-secretase inhibitor, f prolactin thus inactivating Notch signaling. This treatment led to pituitary tumor reduction, lower f GH prolactin and GH tumor content and a decrease in angiogenesis.
    [Show full text]