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C/EBP ∆Uorf Mice – a Genetic Model for Uorf-Mediated Translational Control in Mammals

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C/EBP ∆Uorf Mice – a Genetic Model for Uorf-Mediated Translational Control in Mammals Aus dem Max-Delbrück-Centrum für molekulare Medizin C/EBP uORF mice – a genetic model for uORF-mediated translational control in mammals Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin von Dr. med. Klaus Wethmar Präsident der Humboldt-Universität zu Berlin Prof. Dr. Dr. h.c. Christoph Markschies Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Andreas Herrmann Gutachter: 1. Prof. Dr. Achim Leutz 2. Prof. Dr. Claus Scheidereit 3. Prof. Dr. Thomas Sommer Tag der mündlichen Prüfung: 28. März 2011 Table of contents Table of contents 2 Zusammenfassung 4 Abstract 5 Dedication 7 List of abbreviations 8 1 Introduction 11 1.1 Translational regulation of protein expression 11 1.1.1 Mechanisms of translational control 11 1.1.2 Translational control by upstream open reading frames 14 1.1.2.1 Variable presence of uORFs in alternative transcripts 15 1.1.2.2 Length, position and initiation codon context 15 1.1.2.3 Upstream ORFs integrate the general translational status of a cell 17 1.1.2.4 Upstream ORF-encoded peptides 18 1.1.2.5 Nonsense-mediated mRNA decay 19 1.1.2.6 Variables affecting the degree of uORF-mediated MCS repression 20 1.2 CCAAT/enhancer binding proteins 21 1.2.1 Family overview 21 1.2.2 Isoform-specific functions of C/EBP transcription factors 24 1.2.3 Upstream ORF-mediated control of C/EBP isoform expression 26 1.3 Aims of the thesis 29 2 Materials and Methods 31 2.1 Mice 31 2.1.1 Generation of C/EBPuORF and C/EBPWT mice 31 2.1.2 Genotyping 32 2.1.3 Peripheral blood glucose test 33 2.1.4 Whole mount staining of mouse mammary glands 33 2.1.5 Blood counts 33 2.1.6 Fluorescence activated cell sorting (FACS) 34 2.1.7 Mouse lipopolysaccharid treatment 35 2.1.8 Mouse embryonic fibroblast (MEF) preparation and proliferation assay 35 2.1.9 Osteoclast cultures and TRACP staining 35 2.1.10 Bone histology and histomorphometry 36 2.1.11 Partial hepatectomy and BrdU labeling 36 2.1.12 Liver histomorphometry 37 2.2 Protein analysis 37 2.2.1 Cell lysis, tissue lysis and immunoblotting 37 2.2.2 Enzyme linked immuno sorbent assay (ELISA) 39 2.3 RNA analysis 39 2.3.1 Real-time polymerase chain reaction (real-time PCR) 39 2.3.2 Microarray expression analysis 41 2 Table of contents 2.4 Various methods 41 2.4.1 Chromatin immunoprecipitation 41 2.4.2 Luciferase reporter assays 43 2.4.3 Statistical analysis 45 3 Results 47 3.1 Basic characterization of C/EBP uORF mice 47 3.2 Loss of uORF-mediated LIP expression in C/EBP uORF mice 51 3.3 Defective osteoclast differentiation and altered bone homeostasis in C/EBP uORF mice 53 3.4 Superactivated acute phase gene expression in C/EBP uORF mice 56 3.5 Proliferative defects during liver regeneration in C/EBP uORF mice 58 3.6 Isoform-specific co-regulation of E2F-controlled cell cycle genes 62 4 Discussion 65 4.1 Proof of principle: uORF-mediated translational control in the mouse 65 4.2 C/EBP translational control – exception or paradigm? 66 4.2.1 Mechanisms of uORF-mediated translational control of C/EBP 68 4.3 Do uORFs control the translation of C/EBP? 71 4.4 Physiological relevance of the C/EBP isoform expression ratio 72 4.4.1 LAP and LIP opposingly regulate the differentiation of osteoclasts 73 4.4.2 Increased C/EBP LAP over LIP isoform ratio alters acute phase response gene expression 75 4.4.3 Isoform-specific cell cycle regulation by C/EBPs 78 4.4.4 The function of C/EBP isoforms during liver regeneration 80 4.5 Concluding remarks and future directions 82 4.5.1 Resolving the bipartite functions of C/EBP transcription factors 82 4.5.2 Defining the role of uORFs in the etiology of disease 84 Supplement 87 Acknowledgements 105 References 106 Erklärung 117 Publikationsliste 118 3 Zusammenfassung Evolutionär konservierte, kleine offene Leserahmen (upstream open reading frames, uORFs) sind translational aktive Kontrollelemente, die bevorzugt in Boten-Ribonukleinsäuren von Schlüsselgenen zur Regulation von Zellwachstum, Proliferation und Differenzierung anzutref- fen sind. Man nimmt an, dass uORFs die Proteinexpression von betroffenen Transkripten durch die Regulierung der ribosomalen Reinitiation an 3´ gelegenen alternativen Initiations- codons kontrollieren. Obwohl die physiologische Relevanz uORF-vermittelter translationaler Kontrolle durch humangenetische Studien nahe gelegt wird, existiert bislang kein experimen- telles Wirbeltiermodel, das diese Vermutung belegt. In dieser Arbeit wurden Mäuse analysiert, die defizient für das uORF Initiationscodon des Transkriptionsfaktors CCAAT/enhancer binding protein (C/EBPuORF) sind. Protein- analysen verschiedener Gewebe zeigten, dass C/EBPuORF Mäuse im Gegensatz zu Wildtyp- tieren nicht in der Lage sind, die kurze, auto-antagonistische C/EBP LIP Isoform zu induzie- ren. Die verminderte LIP Expression verursachte eine gestörte Differenzierung knochen- abbauender Osteoklasten und ging mit einer Zunahme von mineralisiertem Knochengewebe in C/EBPuORF Mäusen einher. Nach partieller Hepatektomie führte der Verlust der uORF- vermittelten Induktion von LIP in regenerierenden C/EBPuORF Lebern zu einer Überaktivie- rung C/EBP-regulierter Akute Phase Gene. Im Vergleich zum Wildtyp wiesen Hepatozyten von C/EBPuORF Tieren zudem einen verzögerten und abgeschwächten Wiedereintritt in die S-Phase des Zellzyklus auf. Genomweite Genexpressionsanalysen zeigten, dass die vermin- derte S-Phase Aktivität in regenerierenden C/EBPuORF Lebern mit einer persistierenden Repression von Zellzyklusgenen korrelierte, wobei insbesondere die verminderte Expression zahlreicher E2F-regulierter Gene auffällig wurde. Chromatinimmunpräzipitations- und Repor- tergenexperimente führten zur Entwicklung eines mechanistischen Modells, das eine iso- formspezifische C/EBP-Koregulation E2F-kontrollierter Zellzyklusgene vorschlägt. Die Analyse der C/EBPuORF Mäuse belegt erstmals die Funktionalität der uORF-gesteuerten translationalen Kontrolle im Säugetier. Zusammen mit Sequenzanalysen, welche die weite Verbreitung von uORFs im menschlichen Transkriptom belegen, weisen die hier vorgestellten experimentellen Daten auf eine entscheidende Bedeutung dieses Kontrollmechanismus bei zahlreichen physiologischen und pathopysiologischen Prozessen hin. 4 Abstract Evolutionary conserved small upstream open reading frames (uORFs) are translational control elements predominantly prevalent in the 5’ mRNA regions of key regulatory genes of growth, proliferation, and differentiation. Small uORFs are considered to regulate protein expression by controlling translation re-initiation at downstream initiation codons. Although the physio- logical relevance of uORF-mediated translational control is implied by human genetic studies, no experimental vertebrate model exists to prove this assumption. This thesis comprises the evaluation of mice deficient for the uORF initiation codon of the transcription factor CCAAT/enhancer binding protein (C/EBPuORF). Protein analysis of various tissues demonstrated that C/EBPuORF mice, in contrast to wildtype control animals (C/EBPWT), fail to induce translation of the truncated, auto-antagonistic C/EBP LIP iso- form. The reduced expression of LIP was associated with impaired differentiation of bone resorbing osteclasts and resulted in an increased bone volume of C/EBPuORF mice. After partial hepatectomy the loss of uORF-mediated LIP induction resulted in super activation of acute phase response genes in regenerating livers. Furthermore, C/EBPuORF hepatocytes showed a delayed and blunted re-entry into the cell cycle after partial hepatectomy as com- pared to C/EBPWT animals. Genome-wide transcript expression analyses revealed that the reduced S-phase activity in regenerating C/EBPuORF livers correlated with a persistent re- pression of cell cycle regulatory genes and showed a remarkable underrepresentation of genes regulated by the E2F family of transcription factors. Chromatinimmunoprecipitations and luciferase reporter gene assays allowed the development of a mechanistic model that suggests C/EBP isoform-specific co-regulation of E2F-controled cell cycle genes. The analysis of C/EBPuORF mice validates the functionality of uORF-mediated translational control in vertebrates. Together with sequence analyses that demonstrate the widespread prevalence of uORFs in the human transcriptome, the experimental data presented in this the- sis suggest a comprehensive role of uORF regulation in physiology and the etiology of dis- ease. 5 Dedication for Anne, Klaus, Christine and Lauren. 7 List of abbreviations1 °C degree Celsius A adenosine AML acute myeloid leukemia APR acute phase response APS ammonium persulphate ATP adenosinetriphosphate BCR-ABL breakpoint cluster region - Abelson murine leukemia viral oncogene 1 BMM bone marrow derived macrophage precursors BrdU 5-Bromo-2-deoxy-Uridine bZIP basic leucine zipper C cytosine C. elegans Caenorhabditis elegans C/EBP CCAAT/enhancer-binding protein CD cluster of differentiation CDDO 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid cDNA copy DNA ChIP chromatin immunoprecipitation cm centimeter CPE cytoplasmic polyadenylation element d day DAPI 4',6-diamidino-2-phenylindole ddwater double-distilled deionized water DMEM Dulbecco´s modified Eagles medium DMSO dimethylsulphoxide DNA deoxyribonucelicacid dNTP deoxyribonucleotide DTT dithiothreitol E. coli Escherichia coli ECL enhanced chemiluminescence EDTA ethylenediaminetetraacetate ELISA enzyme linked immunosorbent assay ext
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