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REGULATION OF CAT-1 GENE TRANSCRIPTION DURING PHYSIOLOGICAL AND PATHOLOGICAL CONDITIONS by CHARLIE HUANG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Maria Hatzoglou Department of Nutrition CASE WESTERN RESERVE UNIVERSITY MAY 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. This workis dedicated to my parents (DICK and MEI-HUI), brother (STEVE), and sister (ANGELA) for their love and support during my graduate study. iii TABLE OF CONTENTS Dedication iii Table of Contents iv List of Tables vii List of Figures viii Acknowledgements ix List of Abbreviations xi Abstract xv CHAPTER 1: INTRODUCTION Amino acids and amino acid transporters 1 System y+ transporters 2 Physiological significance of Cat-1 6 Gene transcription in eukaryotic cells 7 Cat-1 gene structure 10 Regulation of Cat-1 expression 11 The Unfolded Protein Response (UPR) and Cat-1 expression 14 Objective of the thesis 20 CHAPTER 2: SP1 REGULATES THE CAT-1 TATA-LESS PROMOTER INTRODUCTION 21 MATERIALS AND METHODS 21 Cell culture and DNA transfection 21 Plasmid constructs 22 iv Electrophoretic mobility shift assay (EMSA) 23 Chromatin immunoprecipitation (ChIP) analysis 23 RT (reverse transcriptase)-PCR and quantitative real time RP-PCR 24 (qRT-PCR) Other methods 24 RESULTS 24 The region from -63 to -25 of the Cat-1 gene promoter is essential for 24 basal expression Sp1 binds the Cat-1 minimal promoter element both in vitro and in 27 vivo The minimal promoter is regulated by ATF4 that binds the AARE in 31 the first exon of the Cat-1 gene DISCUSSION 34 CHAPTER 3: A BIFUNCTIONAL INTRONIC ELEMENT REGULATES THE EXPRESSION OF THE ARGININE/LYSINE TRANSPORTER CAT-1 VIA MECHANISMS INVOLVING THE PURINE-RICH ELEMENT BINDING PROTEIN A (PUR) INTRODUCTION 37 MATERIALS AND METHODS 39 Cell culture and DNA transfection 39 Plasmid constructs 40 Chromatin immunoprecipitation (ChIP) analysis 41 DNA affinity pull-down assays 41 v RT (reverse transcriptase)-PCR and quantitative real time RP-PCR 42 (qRT-PCR) Other methods 42 RESULTS 42 The first intron of the Cat-1 gene contains an enhancer element 41 Identification of Pur as a potential INE-binding protein 46 ATF4 and CHOP regulate INE-mediated Cat-1 gene expression 54 during ER stress Attenuation of Cat-1 transcription during late ER stress requires 57 CHOP DISCUSSION 61 CHAPTER 4: SIGNIFICANCE AND FUTURE PERSPECTIVES 66 APPENDIX 71 BIBLIOGRAPHY 77 vi LIST OF TABLES Table Page 1-1 Amino acid transport systems of mammalian cells 3 1-2 Tissue distribution and transport characteristics of expressed CAT 5 transporters 1-3 Core promoter elements and consensus sequence 9 3-1 WT and MUT (INE) biotinylated oligonucleotides 43 3-2 Sequences of RT-PCR and ChIP primers and siRNA 44 3-3 Identification of proteins associated with the WT and MUT (INE) by 50 mass spectrometry vii LIST OF FIGURES Figure Page 1-1 The Unfolded Protein Response 16 2-1 A GC-rich motif within the Cat-1 gene promoter is required for 26 transcription 2-2 An Sp1 binding site in the Cat-1 gene promoter is required for efficient 28 transcription 2-3 Sp1 binding is required for induction of Cat-1 transcription during amino 32 acid starvation mediated by the AARE and ATF4 3-1 Identification of a regulatory element in the first intron of the Cat-1 gene 47 3-2 Pur binds the INE to increase Cat-1 gene transcription 52 3-3 ATF4 and CHOP bind to the Cat-1 INE in vivo and modulate 55 transcription 3-4 Inhibition of Cat-1 transcription via the INE during late ER stress 58 requires CHOP 3-5 Working model of the role of the INE in the regulation of the Cat-1 gene 64 transcription during ER stress viii ACKNOWLEDGEMENTS I am very grateful and fortunate to have Dr. Maria Hatzoglou as my PhD advisor. I appreciate her positive comments and outlooks when my experiments didn’t turn out the way we anticipated and her patience and understanding at the time when I was dealing with personal issues. If not for her excellent mentorship, outstanding scientific guidance, and unreserved support, I probably would not be what I am today. Her commitment and enthusiasm toward her research will serve as a model for me for my future career. I hope I can become the person she envisions and make her proud. I appreciate the time and the effort Dr. Cheng-Ming Chiang has spent to help me troubleshoot with my experiments. I appreciate the comments, feedbacks, and assistance from Dr. Martin Snider in preparation of our manuscripts and figures. I would like to express my deepest gratitude to Drs. Danny Manor, Edith Lerner, Jonathan Whittaker, and Martin Snider for their valuable time serving on my committee and their insightful comments in helping my research projects progress. I am thankful to former and present members of Hatzglou’s lab, Agata Toborek, Alex Lopez, Calin-Bogdan Chiribau, Celvie Yuan, Chuanping Wang, Dawid Krokowski, Elena Bevilacqua, Francesca Gaccioli, Haiyan Liu, James Fernandez, Lingyin Zhou, Manas Manity, Mithu Majumder, and Yi Li, for their encouragement and support. It ix would have been impossible to accomplish this work without their intellectual and experimental contributions. Thank you all for providing a pleasant environment to do research. Lastly, I am grateful for the unconditional love, the support, and the understanding of my parents, brother, and sister. Your presence is the driving force for my success and accomplishment. NOTE: Chapter 3 of this thesis was originally published in Journal of Biological Chemistry and presented in their entirety. Huang, C. C., Chiribau, C. B., Majumder, M., Chiang, C. M., Wek, R. C., Kelm, R. J., Jr., Khalili, K., Snider, M. D., and Hatzoglou, M. A bifunctional intronic element regulates the expression of the arginine/lysine transporter Cat-1 via mechanisms involving the purine-rich element binding protein A (Pur alpha). J. Biol. Chem. 2009; 284, 32312-32320. © the American Society for Biochemistry and Molecular Biology. x LIST OF ABBREVIATIONS AARE Amino Acid Response Element ARE AU-Rich Element AS Asparagine Synthase ATF Activating Transcription Factor BCL2 B-Cell Lymphoma 2 Bax Bcl-2–associated X Bak Bcl-2 homologous antagonist/killer BIM BCL-2-interacting mediator of cell death BiP Immunoglobulin heavy chain-binding protein BRE TFIIB-recognition element bZIP basic Leucine Zipper Cat Cationic Amino Acid Transporter CATs Cationic Amino Acid Transporters CBP CREB binding protein C/EBP CAAT/Enhancer Binding Protein ChIP Chromatin Immunoprecipitation CHOP CEBP Homology Protein CMV Cytomegalovirus CRE cAMP-response element CS Calf Serum DCE Downstream core element DMEM Dulbecco’s Modified Eagle’s Medium xi DPE Downstream promoter element DTT Dithiothreitol EDEM ER degradation enhancing alpha mannosidase-like EDTA Ethylenediamine Tetraacetic Acid eIF Eukaryotic Translation Initiation Factor EMSA Electrophoretic Mobility Shift Assay ER Endoplasmic Reticulum ERAD Endoplasmic Reticulum Associated Degradation ERp57 Endoplasmic reticulum stress protein 57 ERp72 Endoplasmic reticulum stress protein 72 FBS Fetal Bovine Serum GADD Growth Arrest and DNA Damage-Inducible Protein GAPDH Glyceraldehyde-3-phosphate Dehydrogenase Gcn2p General Control Non-derepressable 2 protein GCN4 General Control Non-derepressable 4 GRP94 Glucose-regulated protein of 94 kDa GTF General transcription factor HDAC1 Histone deacetylase 1 hnRNP heterogeneous nuclear Ribonuclear Protein INE Intronic enhancer element iNOS Inducible nitric oxide synthase Inr Initiator IRE1 Inositol-requiring enzyme 1 xii IRES Internal Ribosome Entry Sequence ITAF IRES Trans-acting Factor KLF Kruppel-like factor LAP Liver-enriched transcriptional activating protein LIP Liver-enriched transcriptional inhibitory protein LUC Firefly Luciferase kb kilobase MEF Mouse Embryonic Fibroblast MTE Motif ten element nNOS Neuronal nitric oxide synthase NO Nitric Oxide ORF Open Reading Frame PCR Polymerase Chain Reaction PDI Protein disulfide isomerase PERK PKR-like Endoplasmic Reticulum Kinase PIC Preinitiation complex PKU Phenylketonuria PTB Polypyrimidine Binding Protein Pur Purine-rich element binding protein alpha Pur Purine-rich element binding protein beta rLUC Renilla Luciferase RNAP II RNA polymerase II rRPL27 Ribosomal protein L27 xiii RT-PCR Reverse Transcriptase-dependent PCR SLC7 Solute Carrier Family 7 Sp Specificity Protein SWI/SNF Switch/Sucrose nonfermentable TAF TBA-associated factors TBP TATA box binding protein TF Transcription factor Tg Thasigargin TRAF2 TNF receptor-associated factor 2 TRB3 Tribbles-related protein 3 uORF upstream ORF UPR Unfolded Protein Response UTR Untranslated Region WT WildType XBP1 X-box binding protein 1 YB-1 Y-box protein 1 xiv Regulation of Cat-1 Gene Transcription during Physiological and Pathological Conditions Abstract by CHARLIE HUANG Expression of the arginine/lysine transporter Cat-1 is highly induced in proliferating and stressed cells via mechanisms that include transcriptional activation.
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