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East Tennessee State University Digital Commons @ East Tennessee State University Electronic Theses and Dissertations Student Works 5-2012 Prelamin A Influences a Program of Gene Expression In Regulation of Cell Cycle Control Christina N. Bridges East Tennessee State University Follow this and additional works at: https://dc.etsu.edu/etd Part of the Biological Phenomena, Cell Phenomena, and Immunity Commons Recommended Citation Bridges, Christina N., "Prelamin A Influences a Program of Gene Expression In Regulation of Cell Cycle Control" (2012). Electronic Theses and Dissertations. Paper 1213. https://dc.etsu.edu/etd/1213 This Dissertation - Open Access is brought to you for free and open access by the Student Works at Digital Commons @ East Tennessee State University. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Digital Commons @ East Tennessee State University. For more information, please contact [email protected]. Prelamin A Influences a Program of Gene Expression in the Regulation of Cell Cycle Control _____________________ A dissertation presented to the faculty of the Department of Biochemistry & Molecular Biology East Tennessee State University In partial fulfillment of the requirements for the degree Doctor of Philosopy in Biomedical Sciences _____________________ by Christina Norton Bridges May 2012 _____________________ Antonio E. Rusiñol, Chair Douglas P. Thewke Sharon E. Campbell Deling Yin Robert V. Schoborg Keywords: Prelamin A, Lamin A, Cell Cycle, Quiescence, Senescence, FoxO, p27, Progeria, Pin1 ABSTRACT Prelamin A Influences a Program of Gene Expression In Regulation of Cell Cycle Control by Christina Norton Bridges The A-type lamins are intermediate filament proteins that constitute a major part of the eukaryotic nuclear lamina—a tough, polymerized, mesh lining of the inner nuclear membrane, providing shape and structural integrity to the nucleus. Lamin A (LA) filaments also permeate the nucleoplasm, providing additional structural support, but also scaffolding numerous tethered molecules to stabilize, organize, and facilitate molecular interactions to accomplish critical functions of cellular metabolism. Over the past 2 decades, much attention has been focused on roles of LA in maintenance of nuclear structural integrity. Only since the late 1990s have scientists discovered the devastating effects of LA gene (LMNA) mutations, as they have associated hundreds of LMNA mutations to a large group of diseases, called laminopathies, with a broad spectrum of phenotypes, ranging from skeletal, muscular, and neurological defects, to defective lipid storage, to accelerated aging phenotypes in diseases called progerias. Recent advances demonstrate LA regulatory functions include cell signaling, cell cycle regulation, transcription, chromatin organization, viral egress, and DNA damage repair. Amidst the flurry of fascinating research, only recently have researchers begun to focus attention on the different isoforms that exist for LA, a precursor form among them. LA is initially synthesized as Prelamin A (PreA), and undergoes a series of modifications that 2 truncate the protein to produce “mature” LA. Existence of the precursor form, and its complex maturation pathway, have puzzled researchers since their realization. With a pattern of expression related to cell cycle phase, we hypothesized a role for PreA in cell cycle control. To investigate, we have performed array studies to assess gene expression effects at the levels of transcript expression, protein expression, and phosphorylation modification status. Here, we present evidence for a PreA-mediated program of cell cycle regulatory gene and protein expression modulation. Implicated pathways include RB-E2F, p53, p27Kip1, FoxOs, p300, and the Cyclins, with additional evidence indicating a role for the Pin1 prolyl isomerase in mediating PreA regulation of the cell cycle. 3 Copyright 2012 by Christina N. Bridges, All Rights Reserved 4 DEDICATION Dedicated to Doris Worley Norton and Mary Jane Charlson To my mother Doris and to Mary Jane, both so beloved to me: Being the recipient of the love, support, and guidance of two such strong, nurturing, beautiful, kind, generous women has been a blessing in my life beyond imagination. The two of you have made me the person that I am, each by showing me the person I want to become. Thank you both, so much, for everything. In memoriam: Doris Worley Norton (September 1, 1955 - August 19, 1992) And as I humbly dedicate everything in my life to God, without headlines or banners, this goes, as well, and I give Him thanks for His many blessings upon me, not the least of which is the gift of the love of my soulmate and husband, Andy, who, by some miracle, has managed to stay with me through these years of the insanity that have been graduate school. Here’s to getting on with life and love… 5 ACKNOWLEDGEMENTS This work was supported by grants from the NIH (1R15HL091502-01A1), East Tennessee State University, and the Progeria Foundation to Antonio Rusiñol and Michael Sinensky. I wish to express my gratitude to Dr. Rusiñol and Dr. Sinensky and these organizations for funding of this work. I would also like to thank the ETSU Biomedical Sciences Graduate Program for funding and support to me. I appreciate Dr. Yaping Zong at Fullmoon Biosciences for providing helpful analysis of data from antibody arrays. Also, I would like to thank Dr. Francis Collins, Director, NIH, and Dr. Robert Goldman, Northwestern University, for providing plasmids used in this project. Grateful Acknowledgement Goes to: Michael Stephan Sinensky: For starting this project, for funding, for amazing pioneering work in the field, and finally, for inspiration. Thank you, Dr. Sinensky! For patience and guidance: My Graduate Committee: Antonio E. Rusiñol, Douglas P. Thewke, Sharon E. Campbell, Robert V. Schoborg, and Deling Yin For thoughtful academic discussion, assistance, and expertise: Michael Sinensky, Philip Musich, and Yue Zou For endless moral support along the way: Mitchell Robinson, Beverly Sherwood, Angela Thompson, Karen Ford, Judy Branson, Brian Rowe, Barbara Turner, Scott Champney, David Johnson, and Jack Rary And for technical assistance and discussions, with a big dose of friendship: Lia Lerner,* Jessica Keasler,* Courtney Netherland, Ben Hilton, Rhesa Dykes, Jaime Parman-Ryans, Theresa Pickle, and Hui Tang *Lia & Jessica performed essential laboratory experiments for this project And finally, my thanks to the Fullerton Genetics Laboratory, Asheville, NC, for allowing me to perform a multitude of sequencing experiments there and for providing me with unbelievable flexibility as an employee while I have pursued this degree, not to mention the compassion and moral support of a wonderful group of friends. 6 CONTENTS Page ABSTRACT ............................................................................................................ 2 DEDICATION .......................................................................................................... 5 ACKNOWLEDGEMENTS ....................................................................................... 6 LIST OF TABLES .................................................................................................... 13 LIST OF FIGURES .................................................................................................. 14 LIST OF ABBREVIATIONS ..................................................................................... 17 Chapter 1. INTRODUCTION ................................................................................................. 23 The Nuclear Lamins ..................................................................................... 23 Posttranslational Lamin Processing ....................................................... 24 The Laminopathies .................................................................................. 26 Lamin A Structural Functions .................................................................. 29 Lamin A Nonstructural Functions ............................................................ 31 Lamin A Functions in Cell Cycle Regulation............................................ 31 Aims of this Study ......................................................................................... 34 2. MATERIALS AND METHODS ............................................................................ 36 Materials ....................................................................................................... 36 General Laboratory Chemicals, Buffers, Reagents ................................. 36 Enzymes, Kits ......................................................................................... 37 General Laboratory Supplies and Equipment.......................................... 38 Materials Used in Cloning/Subcloning and Bacterial Cell Culture ........... 39 Plasmids and Oligonucleotides ............................................................... 39 Cloning Vectors 43 Transfection Reagents ............................................................................ 41 Antibodies ............................................................................................... 41 Mammalian Cell Culture .......................................................................... 42 7 Chapter Page Buffers ..................................................................................................... 42 Software .................................................................................................
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