DEVELOPMENTAL REGULATION OF ELASTIN SYNTHESIS IN CHICKElY AORTA BY TRANSCRIPTIONAL AND POST- TRANSCRIPTIONAL MECHANISMS Yi Hew A thesis submitted in codormity with the requirements for the degree of Doctor of Phiiosophy, Graduate Department of Biochemistry, University of Toronto Copyright by Yin Hew (2001) National Liirary Bibiii ue nationale 1*1 ofcanada du GUIEI9a Acquisitions and Acquisitions et Bibiïïraphic Setvices senrices bibliographiques 3@!5wemlgmÇtieet 395. nw, WNigtOn -ON KlAON4 0ll;rwaON KlAW CYioda Canada The author has granted a non- L'auteur a accordé une licence non exciusive licence ailowhg the exclusive peunettant à la National Li'brary of Canada to Bibliothèque nationale du Canada de reproduce, loan, distriiute or seii reproduire, prêter, diSnliner ou coples of this thesis m microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche//film,de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni Ia thèse ni des extraits substantieis may be printed or otherwise de celle-ci ne doivent être imprimés reproduced withoui the author's ou autrement reproduits sans son permission. autorisation, DEVELOPMENTAL RJZGULATION OF ELASTIN SYNTHESIS IN CHICKEN AORTA BY TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL MECHANiSMS Ph.D. Degree, 200 i Yin Hew Department of Biochemistry, University of Toronto Abstract Synthesis of mit elastin peaks in the perinatal period, and then is strongiy dom- regulated with post-natal vascular deveIopment. Previous studies hmour laboratory have demonstrated a strong cotreIation between this deveIopmenta! pattern of aortic eIastin synthesis and steady state levels of elastin mRNA. In this study the molecular mechanisms by which aortic elastin synthesis is reguiated during normal post-natal deveiopment and growth are elucidated, demonstrating that post-natal dom-regdation of elastin synthesis is due both to decreased tmscription of the elastin gene, and to destabilization of the elastin &NA. Decreased eIastin mRNA stabüity is correlated with protein binding $0 a cis-element in the 3'UTR of elastin. This sequence eIement VGGGGGAGGGAGGGAGGGA), which has been designated the G3A molif; is located within a large, GC-rich region of stable secondary structure in the 3'UTR The G3A motif is conserved across species, and appears to be an important determinant of the secondary structure of the elastin 3'UTR. Protein(s) bindiag to the G3A sequence, are more abundaut in nuclear as compared to cytosolic compartments, and thW presence is associated with tissues producing elastin and correlate with circumstances in which eIastin mRNA is stable. Based on the resuIts of this study, a mode1 is presmted linking the binding of proteins to this G3A site with the structure and stabiIity of the elastin mRNA. Acicnowledgements First of aii, 1wouid iike to thank rny graduate supervisor, Dr Fred Keeley, for ail his encouragement, guidance and unwavering optirnini in my project. He has also given me the fitedom to constantly "try out" new ideas. in addition, 1would aIso like to extend my many thanks to my cornmittee members, Dr Harry Elsholtz and Dr Jm Sodek, who have been extremely helpf'ul and supportive of my work. AU three individuals have been instrumental in my growth and development over the years as a graduate student. 1 would also iike to thank past and present members of my \ab: Eva Sitarz, Julie Heinig, Paul Robson, Zbyszko Grelzczak, CoMie Lau, Cathie Behgham, Xiaojing Dai, Richard Stabl, Dorrie Johnson. They have always been supportive of me in tmsof my work and my personal life. 1would aiso like to especiaiiy thank Zbyszko, Connie and Eva for their excellent technicai contribution to my project. The camaraderie that we have had as a team was wonderhl and precious. 1 will never forget the stimulating discussions we have had as a team. 1 would also Iike to thank other members of the Division of Cardiovascuiar Research, Hospital for Sick ChiIdren whom 1have corne to know and be fiiends with over the years. They include among many others: Susame Holy, Elita Laios, Eric Yang, Claire Coulber, Jamie Lucien, Mingda Shi and Darinka Sakac. Finaiiy, 1wouid like to dedicate this thesis to my beloved family who has given me unconditionai Iove and support throughout the years. To my parents, who have aiways given me the unwavering support and encouragement ever since 1was a child, and has bore with the long distance sepration to enable me to have a graduate education at the University of Toronto. To my sister, Chee, who has aiways been my best fiiend and ally, and who €tas aiways been there for me. Last but not least, to my dearest husoand, Francis, who has aiways beLieved in my abiiities and has encouraged me to be "myself" and to explore what 1tnily want in iife. Coatribntions by other members of the laboratory as üsted by cbapter: Chapter 2 Majority of the initiai primary ceIl cuIture and subsequent subcuItures of the smooth muscle cells were done by Eva Sitan. She was also mainly responsibte for isolation of the aortas fiom the chickens and for the determination of DNA content using the fluorometnc assay. Cbapter 3 Ai1 of the synthesis of the PCR constmcts and subsequent PCR amplification work were performed by Dr Zbyszko Grzelczak. SpecificalIy, he was responsible for the performing reverse transcription of the total RNA preparations and for the PCR amplification experiments to obtain the full-length elastin 3'UTR.. In addition, he also carried out the transformation of pTE2 plasmid and the subsequent restriction digestion of pTE2. Chapter 4 Synthesis of al1 the PCR constmcts used for riboprobe preparation was carried out by Dr Zbyszko Grzelczak. Preparation of protein extracts and nioprobe synthesis were performed in coIIaboration with Connie Lau. Aortic tissue isolation fiom different animals was performed together with Eva Sitan. Chapter 5 Synthesis of ail the PCR constnrcts used for nioprobe preparation was carried out by Dr Zbyszko Grzelczak. Preparation of protein extracts and niprobe synthesis were performed in coIlaboration with Connie Lau. Chapter 6 Isolation of the total RNA was performed in collaboration with Dr Zbyszko Grzelczak. Synthesis of all the constructs for measrrring levels of elastin improcessed and processed RNA; and subsequent PCR amplification work (except for Southern blotting) were performed by Dr Zbyszko Grzelczak- Aortic tissue isolation fiom the older chickens was perfomed together with Eva Sitarz. TABLE OF CONTENTS CHAPTER ONE GENERAL INTRODUCTION.............................................................................. 1 ELASTIC FIBERS ........................................................................................................... 2 Elastogenesis............................................................................................................ 3 Molecular Characterization of Elastin Gene Structure......................................................................... 6 Promoter Region ....................................................................... 7 Sequence and Structure of the Translated Product ................................7 Alternative Splicing of Elastin mRNA .............................................8 Arterial Elastin ... Function and Dlstnbuhon........................................................... 8 Pattern of Synthesis During Development ........................................9 Factors Affecting Elastin Syuthesis.................................................... -10 TRANSCRIPTIONAL REGULATION OF TEE ELASTIN GENE Evidence for Transcriptional Regulation of Elastin Synthesis .................... 12 . Glucocortrcords...................................................................... 12 Rethoic Acids ....................................................................... 13 Growth Factors ....................................................................... 13 Development or TissueSpecifie Signals and Promoter Elements ................ 14 REGULATION OF ELASTIN SYNTHESIS BY mRNA STABILITY Control of mRNA stability in eukaryotes ............................................. IS Sequence or Strwtmd Determinants............................................ 16 Trans-acting Factors.............................................................. -18 Riionucleases....................................................................... 19 Examples of mRNA Stability Determinants and Their Trans-Acting Factors Po& (A) Tail ................................................................ 20 AU-Rich Elements (ME' ................................................. 21 lron Rasponse Element (IRE) of TrddnReceptor mRNA ....... 23 Cytidine-Rich Element (Cmof Globin mRNA ........................ 24 Evidence of Regdation of Elastin by mRNA Stabüity.............................. 26 Pharmacological Agents and Growth Factors ................................... 26 Hypoxia .............................................................................. 27 RATIONALE..................................................................................... -28 CHAPTER TWO ROLE OF mRNA STABILITY LN THE REGULATION OF AORTIC ELASTIN SYNTHESIS DURING DEVELOPMENT AND IN CELL CULTURE .......................... 30 INTRODUCTION ................................................................................... 31 MATERIALS AND METBODS Isolation of Chkken