nm u Ottawa L'Universite canadienne Canada's university run FACULTE DES ETUDES SUPERIEURES l=J FACULTY OF GRADUATE AND ET POSTOCTORALES U Ottawa POSDOCTORAL STUDIES L'Unneisue <anadiemie Canada's university Matias Alvarez-Saavedra AUTEUR DE LA THESE / AUTHOR OF THESIS M.Sc. (Biochemistry) GRADE/DEGREE Department of Biochemistry, Microbiology and Immunology MicroRNA-132-Dependent Post-Transcriptional Regulation of Clock Entrainment Physiology Via Modulation of Chromatin Remodeling and Translational Control Gene Targets TITRE DE LA THESE / TITLE OF THESIS Mary Cheng "WRKTEUTPRE^ CO-DIRECTEUR (CO-DIRECTRICE) DE LA THESE / THESIS CO-SUPERVISOR David Lohnes Adam Rudner Gary W. Slater Le Doyen de la Faculte des etudes superieures et postdoctorales / Dean of the Faculty of Graduate and Postdoctoral Studies MicroRNA-132-Dependent Post-Transcriptional Regulation of Clock Entrainment Physiology Via Modulation of Chromatin Remodeling and Translational Control Gene Targets M.Sc. Thesis in partial fulfillment of the requirements for the degree Master of Science (M.Sc.) in the Biochemistry Program at the University of Ottawa, Faculty of Medicine Ottawa, Ontario, Canada Sliihmil'l-P'rl hv " "J ©Matias Alberto Alvarez-Saavedra, Ottawa, Canada, 2010 1 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 OttawaONK1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-73802-3 Our file Notre r6f6rence ISBN: 978-0-494-73802-3 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduce, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lntemet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la prophete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extra its substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1+1 Canada Thesis Supervisor: Dr. Hai-Ying Mary Cheng, Department of BMI, University of Ottawa Thesis Examiners: Dr. David Lohnes, Department of CMM, University of Ottawa Dr. Adam Rudner, Department of BMI, University of Ottawa Date of Thesis Submission: August 20th, 2010. 2 ABSTRACT Mammalian circadian rhythms of behaviour are synchronized to external time by daily resetting of the master pacemaker, the suprachiasmatic nucleus (SCN), in response to light, in a process known as light-induced clock entrainment. microRNA-132 (miR-132) is induced by light within the SCN via a MAPK-CREB-dependent mechanism and has the capacity to attenuate the entraining effects of light. However, the identity of the genes that miR-132 regulates and their roles in the light-entrainment process have not yet been characterized. This thesis describes that 2 gene clusters involved in chromatin remodeling (i.e. Mecp2, Ep300, Jaridla) and translational control (i.e. Btg2, Paip2a) are under the regulation of miR-132 in the SCN and coordinated regulation of these genes underlies miR132-dependent modulation of mPeriodl (mPerl) and mPeriod2 (mPerl) and the light-entrainment process. I find that the Period genes are bound and transcriptionally modulated by MeCP2. In addition, Paip2a acts as a repressor of Period translation. This work further proposes that miR-132 is enriched for chromatin and translation-associated target genes-and, thus, miR-132 is an important orchestrator of chromatin remodeling and protein translation within the SCN clock, thereby fine-tuning clock entrainment. 3 ACKNOWLEDGEMENTS Dr. Hai-Ying Mary Cheng, my supervisor, for guiding me through the chronobiology field and, very importantly, for financial support. Dr. Odette LaneuviUe, the mediator of this thesis, for constant support through the final stages of this thesis. Dr. David Lohnes, for advice and for being a constant member of my thesis advisory committee from the early stages of this work. Members of the Cheng lab for discussion, in particular Dr. Reynaldo Oliva-Hernandez for help with the mouse colony, tissue harvesting as well as for his friendship and Daniel Cornejo-Palma for help with quantitations and friendship. Dr. Juan I. Young, my previous mentor, for introducing me into the epigenetics field, and for A/ec/?2Floxed/y mice. Many thanks for his friendship and for comments on the revised manuscript (circa March 2010) that arose from this thesis. Dr. Karl Obrietan and Heather Dziema for mouse cannulations and antagomir infusions. Dr. Carolina Perez-Iratxeta for bioinformatics analysis of microRNA-132 targets. Drs. Nahum Sonenberg and Akiko Yanagiya for Paip2a~/~ mice. Dr. Zhaolan (Joe) Zhou for the anti-pS421 MeCP2 antibody. Dr. David Weaver for anti-mPER2, anti-mCLOCK and anti-mBMALl antibodies. Dr. Steven Reppert for anti-mPERl antibody. Dr. Luc Berthiaume for anti-GFP antibodies. Dr. Berge Minassian for h-MeCP2-el-myc vector Dr. Hitoshi Okamura for pcDNA3-mPERl and pBS-mPER2 vectors. Dr. Emilio Lopez-Vidriero, my close friend, for advice. Dr. Rabih Kashouty, my dear friend, for always being there and for believing in me. My family (Victoria, Felipe, Rodrigo & Matias Ignacio) for spiritual and financial support. Sulman Caballero for her unconditional love and spiritual support. My eternal gratitude to Dr. Linda Hsu, Professor Emeritus and my first scientific mentor, for having given me the opportunity to become a researcher during my undergraduate years at Seton Hall University. 4 TABLE OF CONTENTS ABSTRACT 3 ACKNOWLEDGMENTS 4 TABLE OF CONTENTS 5 ABBREVIATIONS 7 INTRODUCTION /. Principles ofcircadian rhythms 10 ii. Genetic basis for circadian rhythmicity 12 Hi. Organization of the circadian system 15 a. The suprachiasmatic nucleus, the master pacemaker 15 b. The retinohypothalamic tract 16 c. Light-induced clock entrainment 17 d. SCN neuroanatomy 19 iv. SCN molecular genetics 22 a. The PAS domain 22 b. The E-box motif. 23 c. The molecular clock 24 d. Human genetics 27 v. Chromatin remodeling and translational control of the clock 28 vi. microRNA function and clock regulation 29 a. microRNA biogenesis 29 b. microRNA regulatory functions 30 c. microRNAs in chronobiology 32 RATIONALE & HYPOTHESIS 34 MATERIALS & METHODS 36 RESULTS 50 /'. Generation & characterization of microRNA-132 transgenic mice 50 ii. Behavioural analyses of microRNA-132 transgenic mice 55 Hi. Bioinformatics analysis of predicted microRNA-132 targets 62 iv. In-vitro analysis of microRNA-132 MREs 82 5 v. In-vitro knockdown ofmicroRNA-132 85 vi. In-vivo knockdown ofmicroRNA-132 in the murine SCN 85 vii. microRNA-132 target analysis in microRNA-132 transgenic mice 87 viii. microRNA-132 target analysis in microRNA-219 transgenic mice 97 ix. microRNA-132 target genes are activity- dependent 98 x. microRNA-132 overexpression 's effects on core clock genes 102 xi. MeCP 2-dependent transcriptional modulation of the Period genes 109 xii. PAIP2A -dependent translational control of the Period genes 117 DISCUSSION 130 i. microRNA-132 target identification 130 ii. microRNA-132 as an orchestrator of chromatin remodeling and translational control in light-induced clock resetting 130 /'/'/. Light-induction of molecular waves that regulate the clock-resetting response: The light-induced molecular waves hypothesis 134 iv. Outlook 138 BIBLIOGRAPHY 140 CONTRIBUTIONS OF COLLABORATORS 162 CURRICULUM VITAE 164 APPENDIX 167 6 ABBREVIATIONS AHR: Aryl Hydrocarbon Receptor ARNT: Aryl Hydrocarbon Receptor Nuclear Translocator AVP: Arginine Vasopressin BMAL1: ARNT-Like Protein 1, Brain and Muscle BTG2: B-Cell Translocation Gene 2 B-TRCP1: Beta-Transducin Repeat-Containing Protein CAR: Calretinin c-FOS: cellular Fos (Finkel-Biskis-Jinkins murine osteogenic sarcoma virus) ChIP: Chromatin Immunoprecipitation CK18: Casein Kinase 1 Delta CK1 •: Casein Kinase 1 Epsilon CLOCK: Circadian Locomotor Output Cycles Kaput CRE: Cyclic-AMP Response Element CREB: c-AMP Response Element Binding Protein CT: Circadian Time DD: Dark:Dark DICER: Helicase with RNA Motif, Drosophila Homolg DOX: Doxycycline dpc: days post-coitum DROSHA: Nuclear Ribonuclease III, Drosophila Homolog DSPS: Delayed Sleep-Phase Syndrome ECFP: Enhanced Cyan Fluorescent Protein ENK: Enkephalin ENU: iV-ethyl-TV-nitrosourea EP300: El A-Binding Protein, 300kD FASPS: Familial Advanced Sleep-Phase Syndrome FBXL3: F-Box and Leucine-Rich Repeat Portein