Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2010 Characterization of the Functional Role of the Intracellular Cholesterol Transporter StARD4 in Knockout Mice, and Investigation of Epigenetic Modulation of ApoA-I Transcription Joshua Riegelhaupt Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Riegelhaupt, Joshua, "Characterization of the Functional Role of the Intracellular Cholesterol Transporter StARD4 in Knockout Mice, and Investigation of Epigenetic Modulation of ApoA-I Transcription" (2010). Student Theses and Dissertations. Paper 70. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. Characterization of the Functional Role of the Intracellular Cholesterol Transporter StARD4 in Knockout Mice, and Investigation of Epigenetic Modulation of ApoA-I Transcription A Thesis presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Joshua Riegelhaupt June 2010 © Copyright by Joshua Riegelhaupt, 2010 CHARACTERIZATION OF THE FUNCTIONAL ROLE OF THE INTRACELLULAR CHOLESTEROL TRANSPORTER StARD4 IN KNOCKOUT MICE AND INVESTIGATION OF THE EPIGENTIC MODULATION OF APOA-I TRANSCRIPTION Joshua Riegelhaupt, Ph.D. The Rockefeller University 2010 Cholesterol is crucial for mammalian survival by playing important roles, such as regulating membrane fluidity and as a precursor for the synthesis of steroid and sex hormones, bile acids, and Vitamin D. In addition, cellular and organismal regulation of cholesterol is important for health. For example, increased levels of plasma LDL cholesterol are a risk factor for coronary heart disease and stroke. Intracellular cholesterol levels are regulated by a variety of mechanisms, but numerous studies indicate a very important role for transcriptional regulation by Sterol Regulatory Binding Proteins (SREBPs), Liver X Receptors (LXRs) and Unfolded Protein Response (UPR) or ER stress families of transcription factors. StARD4 is regulated by SREBPs and we have chosen to make a mouse knockout model to characterize its role in-vivo. StARD4, expressed primarily in liver and macrophages, is a known intracellular cholesterol transporter previously shown to be down-regulated ~2 fold in liver, by high cholesterol feeding. It is thought to be involved in the dynamics of cholesterol movement between ER, plasma membrane, endosomes and lipid droplets. Based on these observations, I hypothesized that a knockout of StARD4 in a mouse model would show altered intracellular cholesterol sorting, and figuring out the basis of such a defect would provide insight into the general mechanisms of intracellular sterol transport. To my surprise, StARD4 knockouts were viable and for the most part phenotypically normal. They showed no alteration in plasma or liver cholesterol or triglycerides. In addition, no abnormalities were found in glucose metabolism, macrophage cholesterol efflux, or atherosclerosis susceptibility. Based on these observations, I hypothesize that in-vivo, the absence of StARD4 is compensated for by other genes and/or pathways. In the future, it will be necessary to identify these compensatory mechanism(s) to truly understand the physiological role of StARD4. I also studied another aspect of cholesterol metabolism related to its transport in plasma in high density lipoproteins (HDLs). HDL is involved in the reverse cholesterol transport mechanism, whereby excess cholesterol is removed from peripheral tissues and transported to the liver for excretion. The major protein of HDL is apoA-I and in mouse models it has been shown that animals transgenic for apoA-I have increased HDL levels. This suggests increased apoA-I transcription as a mechanism for increasing HDL, which might be preventive or therapeutic for coronary heart disease. With this as a goal, as part of my thesis I studied the epigenetic regulation of apoA-I transcription. I found that increased apoA-I transcription in liver cell culture cell lines was associated with highly unmethylated CpGs in the apoA-I promoter, and the reverse, in cultures with poor apoA-I expression. I also found histone marks associated with apoA-I expression. This project was discontinued in favor of the StARD4 knockout mouse project. However, it might be continued in the future to reveal drug targets that alter epigenetic regulation of apoA-I in a manner that raises HDL levels. Acknowledgments I would like to take a moment to thank the many people who have helped me along my path before and during my PhD experience. First and foremost, I would like to extend my deepest gratitude to my PhD mentor, Jan Breslow. Above all else, Jan taught me the virtue of patience and humbleness. I can not think of a kinder or more encouraging person to have been my guide through the ups and downs of the PhD experience. At no point, did Jan lose faith in my ability or intelligence and for this I am greatly appreciative. I am also thankful to the members of team Breslow of which there have been many. It began with my relationship with Marc Waase, who happened to work on similar projects to my own. This was quickly expanded to include my post doc mentors, Ralph Burkhardt, Changcheng Zhou, El Doctor Jose Manuel Rodriquez and Jeanne Garbarino. I also enjoyed the time spent with all other members of the Breslow lab, which included, Brian Pridgen, Megan Roblee, Eimear Kenny and KY Chen. I had the pleasure of working with a Cornell medical student, Daniel Cruz, whose company I greatly enjoyed over the summer we were together. I wanted to thank Helen Yu for all her work sectioning my mouse aortas and Katie Tsang for making the lab run so smoothly. I would also like to thank the two clinical scholars Manish Ponda and Swaroop Pendyala who taught me a lot of things about medical biology that I would not have learned otherwise. I am also grateful to Lauren Glenz who helped run the day to day administrative functions of the laboratory. The Tri-institutions have many useful resources that I was able to use during my time here and I would like to thank the people who I worked with; Suzanna Couto (MSKCC Pathology), Chingewen Yang (RU Gene Targeting), Rada Norinsky (RU Transgenics), Connie Zhao (RU Genomics) and all their staffs. I am also grateful to my classmates, the PhD entering class of 2005, whom I have been able to share the joys and stresses of our time at Rockefeller together. Specifically, I would like to thank my roommate Florian Gehre, for the great times we spent together. My graduate career would not have run as smoothly without the help of everyone behind the scenes in the Deans office at the Rockefeller University. Sid Strickland, Emily Harms, Marta Delgado, Kristen Cullen, Cristian Rosario and Michelle Sherman. I thank my committee members: Sid Strickland and David Allis, for their advice and assistance over the past years. I would also like to thank my external member, David Cohen (Harvard) for his comments and help. Finally, I would like to thank my family. My parents, Elliot Riegelhaupt and Susan Steinberg are much of the influence that has iii gotten me here today and without their constant love and support, I would not be who I am. Their hardwork and concomitant family devotion is a wonderful reminder of what it takes to be successful in this world. My brothers Paul and Mike and sister Rachel always provide a great friendship which I can rely on no matter where I am in life. My grandparents, Lillian and Milton are people who I look up to in admiration for the struggles they have endured in life and for the successes they have achieved. My aunt and uncle, Lu and Michael and my cousins, Ilana and Talia, are wonderful support networks for me. Finally, my girlfriend Sasha Growick, who had the pleasure of being with me for almost all of my time at Rockefeller. Thanks for all your love and support. I could not have done it without you. iv Table of Contents Acknowledgments .............................................................................iii List of Figures......................................................................................ix List of Tables .......................................................................................xii List of Abbreviations ........................................................................xiv Chapter 1: Introduction Cholesterol’s Physiological Importance ...............................................1 Cholesterol de novo Synthesis and Dietary Uptake ........................4 Lipoprotein Transport for Cholesterol in the Blood .......................6 Studying Cholesterol Metabolism and Atherosclerosis in a Mouse Model .........................................................................................................8 The HDL Lipoprotein......................................................................10 HDL Components: ApoA-I & ApoA-II..........................................11 Lipid Acquisition and Particle Maturation......................................12 HDL Catabolism: SR-BI & CETP ..................................................14 Transcriptional Control of ApoA-I .................................................16 Epigenetics ...........................................................................................19