THE FUNCTIONAL STUDY OF TRANSCRIPTIONAL COREPRESSOR G- PROTEIN SUPRESSOR 2 (GPS2) AND TUMOR SUPRESSOR PROMYELOCYTIC LEUKEMIA (PML) by XIWEN CHENG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Dr. Hung-Ying Kao Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY August, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of __Xiwen Cheng__________________ candidate for the Ph.D. degree*. (signed)_______David_Samols__Ph.D._________________ (chair of the committee) _____________Edward Stavnezer__Ph.D._____________ _____________Gary Landreth___Ph.D.________________ _____________Hung-Ying Kao___Ph.D.________________ _____________Youwei Zhang____Ph.D._______________ ________________________________________________ (date) __May 26, 2010____________ *We also certify that written approval has been obtained for any proprietary material contained therein. To my parents and family Table of Contents LIST OF TABLES ................................................................................................. 7 LIST OF FIGURES ............................................................................................... 8 ACKNOWLEDGEMENTS ................................................................................... 12 LIST OF ABBREVIATIONS ................................................................................ 13 CHAPTER 1: INTRODUCTION ...................................................................... 19 Transcription: a central mechanism to maintain the normality of life.................................................................................................................... 19 Nuclear receptors (NRs): a family of sensors to hormonal signaling .......................................................................................................... 21 Transcription cofactors: coactivators and corepressors .................................. 22 SMRT corepressor complexes and GPS2 ....................................................... 25 The identification of GPS2 ............................................................................... 27 GPS2 interacts with many cellular proteins ..................................................... 28 The biological functions of GPS2 .................................................................... 29 The structure, motif and signature sequence of GPS2 protein ........................ 34 Estrogen receptor and breast cancers ............................................................ 35 The tamoxifen-resistant breast cancers .......................................................... 37 Acute promyelocytic leukemia (APL) ............................................................... 38 The promyelocytic leukemia protein (PML) ..................................................... 41 PML nuclear bodies......................................................................................... 42 PML: an emerging tumor suppressor .............................................................. 43 PML plays a role in endothelial physiology ...................................................... 43 1 PML is induced by inflammation ...................................................................... 44 Angiogenesis, tumor growth and inflammation ................................................ 45 The controversy of TNFα in tumor therapy ...................................................... 46 Microarray is a powerful tool to identify alterations of expression of genes at genomic scale ............................................................................... 50 CHAPTER 2: G-PROTEIN PATHWAY SUPRESSOR 2 (GPS2), AN INTEGRAL COMPONENT OF THE SMRT COMPLEXES, IS IMPORTANT FOR ESTROGEN RECEPTOR ALPHA (ERα)- MEDIATED TRANSCRIPTIONAL REGULATION AND CELL PROLIFERATION ............................................................................................... 54 ABSTRACT ..................................................................................................... 54 INTRODUCTON .............................................................................................. 55 MEATERIALS AND METHODS ...................................................................... 58 Reagents, plasmids, and antibodies ............................................................ 58 Affinity Antibody purification ......................................................................... 59 Yeast two-hybrid screens ............................................................................. 60 GST pull-down assays ................................................................................. 60 Co-immunoprecipitation of transfected protein ............................................ 61 Histone deacetylase assays ........................................................................ 62 Fluorescence microscopy ............................................................................ 62 Subcellular fractionation ............................................................................... 63 Quantitative reverse transcription PCR (qRT-PCR) ..................................... 63 Transient transfection of siRNA ................................................................... 64 2 Luciferase reporter assays ........................................................................... 64 Chromatin immunoprecipitaion (ChIP) assays ............................................. 65 RESULTS ........................................................................................................ 69 Isolation of GPS2 as a SMRT-interaction protein by Yeast two- hybrid (Y2H). ................................................................................................ 69 GPS2 binds to SMRT in vivo and in vitro ..................................................... 69 GPS2 associates with histone deacetylases. ............................................... 71 GPS2 is a transcriptional corepressor. ......................................................... 71 GPS2 associates with ERα target gene promoter in a ligand- dependent manner ....................................................................................... 73 Knockdown of GPS2 altered the expression of ERα target genes. .......................................................................................................... 74 Knockdown of GPS2 altered the pattern of recruitment of SMRT corepressor complex components .................................................... 75 GPS2 and SMRT suppress breast cancer cell proliferation. ........................ 77 DISCUSSION ................................................................................................ 101 Biological significance of the study ............................................................ 101 The role of GPS2 in transcriptional regulation. .......................................... 102 The role of GPS2 in ERα-mediated transcription. ...................................... 103 Recruitment of corepressors to ERα target gene promoter. ....................... 105 The role of corepressor complex in breast cancers. .................................. 105 CHAPTER 3: PROMYELOCYTIC LEUKEMIA PROTEIN (PML) REGULATES ENDOTHELIAL CELL NETWORK FORMATION 3 AND MIGRATION IN RESPONSE TO TUMOR NECROSIS FACTOR ALPHA (TNFα) AND INTERFERON ALPHA (IFNα) ......................... 109 ABSTRACT ................................................................................................... 109 INTRODUCTION ........................................................................................... 110 MATERIALS AND METHODS ...................................................................... 113 Reagents and Antibodies ........................................................................... 113 Cell culture, Drug treatment and siRNA transfection .................................. 114 Total RNA extraction, RT-PCR and Real-time PCR ................................... 114 Immunofluorescence microscopy............................................................... 115 Promoter analysis ...................................................................................... 115 Chromatin immunoprecipitaion (ChIP) assays ........................................... 116 In vitro angiogenesis assay ........................................................................ 117 In vitro wound-healing assay ..................................................................... 117 Western blotting and quantification of band intensity ................................. 118 RESULTS ...................................................................................................... 119 DISCUSSION ................................................................................................ 140 CHAPTER 4: MICROARRAY ANALYSIS OF THE TRANSCRIPTOME FROM ENDOTHELIAL CELLS FOLLOWING PML KNOCKDOWN ......................................................................................... 146 ABSTRACT ................................................................................................... 146 INTRODUCTION ........................................................................................... 147 MATERIALS AND METHODS ...................................................................... 152 Sample preparation and the arrays ............................................................ 152 4 Data preprocessing ...................................................................................