Transcriptional regulation by the Wilms' tumor suppressor gene (WT1)

다국어 초록 (Multilingual Abstract)

Wilms' tumor (WT) is an embryonic renal neoplasm which affects 1 in 10,000 children. It has long been considered an excellent model for studying the relationship of cancer to development. A tumor suppressor gene wt1, implicated in predisposition to WT, is known to play an essential role in regulating development of the kidney and of the genitourinary system. A number of genes involved in growth regulation and cellular differentiation are modulated by WT1 and a number of proteins are known to associate with WT1. Some of these proteins are able to alter the transcriptional properties of WT1 influencing whether WT1 acts as a repressor or activator.
We demonstrate that WT1 represses different classes of activation domains previously shown to stimulate the initiation and elongation steps of transcription in vivo. We also showed that WT1 can repress transcription over a significant distance. Nuclear run-on assays revealed that the mechanism of repression by WT1 occurs at the level of transcription initiation.
We identified a novel WT1-interacting protein named Bone Marrow Zinc Finger 2 (BMZF2). The BMZF2 gene encodes a potential transcription factor harboring 18 zinc fingers and mainly expressed in fetal tissues. In vivo and in vitro pull-down experiments showed that WT1 and BMZF2 associate. This interaction inhibits WT1-mediated transcriptional activation. Additionally, BMZF2 harbors a transcriptional repression domain. These results suggest that BMZF2 interferes with the transactivation properties of WT1.
We performed an expression array screen utilizing WT1 inducible cell lines and found the human vitamin D receptor (VDR) as a major target. Nuclear run-on experiments, transient transfection assays, deletion mutagenesis, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays suggested that the WT1(-KTS) isoforms bind directly to the VDR promoter and activate VDR gene expression. Our results suggest that the human VDR gene represents a downstream target of WT1. We discuss the potential regulation of VDR by WT1 in normal and malignant tissues.

다국어 초록 (Multilingual Abstract)

La tumeur de Wilms (WT) est un ne´oplasms re´nal embryonnaire qui affecte 1 enfant sur 10 000. Elle a longtemps e´te´ conside´re´e comme un excellent mode｀le pour l'e´tude de la relation entre le cancer et son de´veloppement. Le ge｀ne suppresseur de tumeurs wt1, implique´ dans la pre´disposition a｀ la WT, est reconnu pour jouer un ro＾le essentiel dans la re´gulation de de´veloppement du rein et du syste｀me uroge´nital. Certains ge｀nes implique´s dans la re´gulation de la croissance et dans la diffe´rentiation cellulaire sent module´s par la prote´ine WT1 et quelques autres prote´ines peuvent s'y associer. Certaines de ces prote´ines sont capables d'alte´rer les proprie´te´s de transcription de WT1 influenc¸ant cette dernie｀re a｀ agir soit comme un re´presseur ou soit comme un activateur.
Nous avons de´montre´ que WT1 inhibe diffe´rentes classes de domains d'activation reconnus pour stimuler les e´tapes d'initiation et d'e´longation de la transcription in vivo. Nous avons e´galement de´montre´ que WT1 peut inhiber la transcription sur une distance significative. Nos donne´es indiquent que WT1 peut influencer l'activite´ d'activateurs qui fonctionne en stimulant l'initiation ou l'e´longation.
Nous avons aussi identifie´ une nouvelle prote´ine interagissant avec WT1 appele´e BMZF2 (Bone Marrow Zinc Finger 2). Le ge｀ne BMZF2 code pour un facteur de transcription potentiel qui comports 18 doigts do zinc et qui s'exprime principalement dans les tissus fe´taux. Par chromatographie d'affinite´ et immunopre´cipitation, nous avons montre´ que WT1 et BMZF2 sont associe´s in vitro et in vivo. Cette interaction inhibe l'activation de la transcription me´diee par WT1. De plus, BMZF2 contient un domaine de re´pression de la transcription. Ces re´sultats sugge｀rent que BMZF2 interfe｀re avec les proprie´te´s de transactivation de WT1.
En criblant une banque d'expression de ligne´es cellulaires inductibles par WT1 nous avons trouve´ que le re´cepteur humain de la vitamine D (VDR) en e´tait la cible principale. Les analyses de la re´gulation en position cis de WT1 (nuclear run-on), les essais de transfectants stables, la mutage´ne｀se par de´le´tion, les essais de changement de la mobilite´ e´lectrophore´tique et ceux d'immunopre´cipitation de la chromatine sugge｀rent que les isoformes de WT1 (-KTS) sont associe´s directement au promoteur do VDR et activent l'expression de ge｀ne VDR. Nos re´sultats indiquent que le ge｀ne humain VDR repre´sente une cible pour WT1. Finalement, nous discutons le potentiel de re´gulation de VDR par WT1 dans les tissus normaux et cance´reux.

목차 (Table of Contents)

• ABSTRACT = ii
• RE´SUME´ = iii
• PREFACE = v
• TABLE OF CONTENTS = vii
• LIST OF FIGURES = xi
• LIST OF TABLES = xiii
• LIST OF ABBREVIATIONS = xiv
• Original Contributions to Knowledge = xv
• ACKNOWLEDGEMENTS = xvi
• CHAPTER 1 General Introduction = 1
• 1-1. Wilms' tumor = 2
• 1-2. Structure of the Wilms' tumor suppressor gene (WT1) mRNA and protein = 4
• 1-3. Wilms' tumor associated syndromes = 11
• 1-4. WT1 in kidney development = 13
• 1-5. WT1 and sex determination = 15
• 1-6. EWS-WT1 fusion protein in DSRCT = 18
• 1-7. Regulation of the WT1 expression = 19
• 1-5-1. Transcriptional regulation = 19
• 1-3-4-1. PAX2 and PAX8 = 22
• 1-3-4-2. Sp1 = 22
• 1-3-4-3. GATA-1 = 23
• 1-3-4-4. Complex D = 23
• 1-3-4-5. WT1 = 23
• 1-5-2. Phosphorylation = 23
• 1-8. Candidate WT1 downstream target genes = 24
• 1-9. WT1 interacting proteins = 29
• 1-9-1. p53, p73, and p63 = 29
• 1-9-2. Prostate Apoptosis Response-4 Protein (Par-4) = 31
• 1-9-3. CIAO1 = 31
• 1-9-4. U2AF65 = 32
• 1-9-5. Human Ubiquitin-Conjugating Enzyme 9 = 32
• 1-9-6. Meat Shock Protein 70 = 33
• 1-9-7. WTAP = 33
• 1-9-8. CBP/P300 = 33
• 1-10. Conclusions = 34
• CHAPTER 2 The Wilms' tumor suppressor gene (wt1) product represses different functional classes of transcriptional activation domains = 35
• 2-1. ABSTRACT = 36
• 2-2. INTRODUCTION = 36
• 2-3. MATERIALS AND METHODS = 38
• 2-3-1. Plasmid construction = 38
• 2-3-2. Cell lines, transfections, and CAT assays = 40
• 2-3-3. Nuclear run-on analysis = 41
• 2-4. RESULTS = 42
• 2-4-1. WT1 can repress different functional classes of activators = 42
• 2-4-2. The N-terminal domain of WT1 is required for repression of type IIB activators = 51
• 2-4-3. Nuclear run on analysis of WT1 inhibition of initiation = 56
• 2-5. DISCUSSION = 62
• 2-6. ACKNOWLEDGEMENTS = 64
• CHAPTER 3 Inhibition of Wilms' tumor 1 (WT1) transactivation by bone marrow zinc finger 2, a novel transcriptional repressor = 65
• 3-1. ABSTRACT = 66
• 3-2. INTRODUCTION = 66
• 3-3. MATERIALS AND METHODS = 68
• 3-3-1. Materials and General Methods = 68
• 3-3-2. Cell culture, transfections, and CAT and luciferase assays = 69
• 3-3-3. Plasmid construction = 69
• 3-3-4. Preparation of Hela Nuclear Extracts = 72
• 3-3-5. Isolation of BMZF2 by affinity chromatography = 72
• 3-3-6. Native Co-Immunoprecipitations of BMZF2 and WT1 = 73
• 3-3-7. Northern blotting = 74
• 3-3-8. Subcellular localization of BMZF2 and WT1 = 75
• 3-3-9. In vitro transcription and translation = 75
• 3-3-10. GST Pulldown assays = 76
• 3-3-11. Immunoprecipitation assays = 76
• 3-3-12. Nucleotide sequence accession number = 71
• 3-4. RESULTS = 77
• 3-4-1. Isolation of BMZF2 as a novel WT1-interacting protein = 77
• 3-4-2. In vivo and in vitro interaction between BMZF2 and WT1 = 82
• 3-4-3. Expression of BMZF2 mRNA in human tissues and subcellular localization of BMZF2 = 90
• 3-4-4. Inhibition of WT1-mediated activation by BMZF2 = 90
• 3-4-5. BMZF2 is a transcriptional repressor = 97
• 3-5. DISCUSSION = 100
• 3-6. ACKNOWLEDGEMENTS = 103
• CHAPTER 4 Functional characterization of WT1 binding sites within the human vitamin D receptor gene promoter = 104
• 4-1. ABSTRACT = 105
• 4-2. INTRODUCTION = 105
• 4-3. MATERIALS AND METHODS = 108
• 4-3-1. Materials and General Methods = 108
• 4-3-2. Piasmid construction = 108
• 4-3-3. Cell culture, transfection and luciferase assay = 109
• 4-3-4. cDNA array analysis = 110
• 4-3-5. S1 nuclease analysis and Western blotting = 110
• 4-3-6. Electrophoretic mobility shift assays (EMSAs) = 112
• 4-3-7. Nuclear run-on analysis = 113
• 4-3-8. Chromatin immunoprecipitation(ChIP) = 113
• 4-4. RESULTS = 114
• 4-4-1. Expression Profiling in 293/WT(+/-) cells for genes under WT1 regulation = 114
• 4-4-2. Identification of the WT1-responsive sites in the human VDR promoter = 124
• 4-4-3. Transcriptional activation of the VDR promoter by WT1 = 127
• 4-5. DISCUSSION = 136
• 4-6. ACKNOWLEDGEMENTS = 139
• CHAPTER 5 General discussion = 140
• CHAPTER 6 References = 151