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Understanding the Role of E2F7 in the Regulation of Cellular Proliferation and DNA Damage Responses

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Understanding the Role of E2F7 in the Regulation of Cellular Proliferation and DNA Damage Responses Understanding the role of E2F7 in the regulation of cellular proliferation and DNA damage responses Jon Vallejo Rodríguez Leioa, 2018 (c)2018 JON VALLEJO RODRIGUEZ Understanding the role of E2F7 in the regulation of cellular proliferation and DNA damage responses PhD Thesis Jon Vallejo Rodríguez Leioa, 2018 Supervisors: Ana María Zubiaga Elordieta and Asier Fullaondo Elordui-Zapaterieche Jon Vallejo Rodríguez was recipient of a research training fellowship from the University of the Basque Country UPV/EHU (PIF2013). This thesis work has been funded by the Spanish Ministry of Economy and Competitiveness (SAF2012-33551 and SAF2015- 67562-R), the Basque Government (IT634-13) and the University of the Basque Country UPV/EHU (UFI11/20). Table of contents TABLE OF CONTENTS ....................................................................................................... 17 ABBREVIATIONS ............................................................................................................... 21 ABSTRACT ........................................................................................................................ 25 1. - INTRODUCTION .......................................................................................................... 27 1.1. E2F family of transcription factors ................................................................................................ 29 1.1.1. Mechanisms of transcriptional regulation by E2F family members ............................................ 31 1.1.2. Regulation of E2F factor expression ............................................................................................ 33 1.1.3. Functions of E2F protein family ................................................................................................... 36 1.1.3.1. Cell cycle regulation by E2F factors ...................................................................................... 36 1.1.3.2. Apoptosis regulation by E2F factors..................................................................................... 39 1.1.3.3. E2F factors and cancer ......................................................................................................... 41 1.2. The DNA Damage Response and E2F............................................................................................. 43 1.2.1. Sources of DNA damage and types of DNA lesions ..................................................................... 43 1.2.1.1. Endogenous sources of DNA damage .................................................................................. 43 1.2.1.2. Exogenous sources of DNA damage ..................................................................................... 44 1.2.2. DNA damage response ................................................................................................................ 45 1.2.2.1. DNA damage sensors and transducers ................................................................................ 45 1.2.2.2. DDR signaling pathways ....................................................................................................... 46 1.2.3. DNA damage and cell cycle control ............................................................................................. 50 1.2.4. Role of E2F factors in the DNA Damage Response. ..................................................................... 51 2.- HYPOTHESIS AND AIMS ............................................................................................ 53 3.- MATERIALS AND METHODS ...................................................................................... 57 3.1 Cellular biology methods ............................................................................................................... 59 3.1.1. Cell lines and culture conditions .................................................................................................. 59 3.1.2. Cell lines generated in this thesis ................................................................................................ 60 3.1.2.1. U2OS-E2F7-KO cells .............................................................................................................. 60 3.1.2.2. U2OS-TRE-E2F1 .................................................................................................................... 62 3.1.3. Transfection ................................................................................................................................. 62 3.1.4. Cell synchronization .................................................................................................................... 63 3.1.5. DNA replication assays (BrDU) .................................................................................................... 63 3.1.6. Proliferation assays ..................................................................................................................... 64 3.1.6.1. Carboxyfluorescein succinimidyl ester (CFSE) assay ............................................................ 64 3.1.6.2. Crystal violet staining ........................................................................................................... 65 3.1.6.3. Colony formation assay ........................................................................................................ 65 3.1.7. Mitotic index analysis and DDR quantification assay .................................................................. 65 3.1.8. Homologous recombination efficiency assay .............................................................................. 66 3.1.9. Analysis of chromosomal aberrations ......................................................................................... 67 3.1.10. High-throughput apoptosis screening assay .............................................................................. 67 3.2. Molecular biology techniques ...................................................................................................... 68 3.2.1. RNA expression analyses ............................................................................................................. 68 3.2.1.1.RNA extraction ...................................................................................................................... 68 3.2.1.2. RT-Q-PCR of mRNA ............................................................................................................... 69 3.2.1.2. RT-Q-PCR of microRNA ......................................................................................................... 71 3.2.2. Protein expression analyses ........................................................................................................ 73 3.2.2.1. Protein extraction ................................................................................................................. 73 3.2.2.1. Western Blotting .................................................................................................................. 73 3.2.3. Chromatin Immunoprecipitation (ChIP) analyses ........................................................................ 75 3.2.4. Co-Immunoprecipitation (CoIP) analyses .................................................................................... 77 3.3. Bioinformatic analyses ................................................................................................................. 77 3.3.1. Localization of E2F consensus motifs .......................................................................................... 77 3.3.2. Identification of protein motifs ................................................................................................... 78 3.4. Statistical analyses ....................................................................................................................... 78 4.- RESULTS ...................................................................................................................... 79 4.0 PREVIOUS FINDINGS GENERATED IN THE LABORATORY ................................................................ 81 4.1 FUNCTIONAL ANALYSIS OF microRNAs REGULATED BY E2F7 ......................................................... 83 4.1.1 Validation of the microRNA profiling ............................................................................................ 83 4.1.2 Regulation of microRNA expression by E2F7 ................................................................................ 84 4.1.2.1 In vivo transcription factor binding to the promoter regions of microRNAs regulated by E2F7 ................................................................................................................................................... 86 4.1.2 Functional activity of E2F7-downregulated microRNAs ............................................................... 88 4.2 ROLE OF E2F7 IN DNA DAMAGE RESPONSE AND REPAIR ............................................................... 93 4.2.1 Characterization of a set of DNA damage response genes as direct E2F7 targets ....................... 93 4.2.2 Role of E2F7 in the cellular responses to genotoxic DNA damage ............................................... 96 4.2.2.1 E2F7 attenuates cell cycle progression upon DNA lesions affecting replication fork progression ........................................................................................................................................ 96 4.2.2.2 E2F7-dependent gene expression regulation of DDR genes upon DNA damage ................ 102 4.2.3 Role of E2F7
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