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Negative Regulation of Gene Expression by the Tumor Suppressor P53

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Negative Regulation of Gene Expression by the Tumor Suppressor P53 Negative regulation of gene expression by the tumor suppressor p53 Anthony M. Barsotti Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2011 © 2011 Anthony M. Barsotti All Rights Reserved ABSTRACT Negative regulation of gene expression by the tumor suppressor p53 Anthony M. Barsotti The tumor suppressor p53 inhibits the expression of a substantial number of genes whose protein products serve to promote cell survival or cell cycle progression, thereby ensuring efficient execution of p53-dependent apoptosis, cell-cycle arrest or senescence. Furthermore, p53-mediated repression has also been shown to participate in pathways that regulate diverse cellular processes, including angiogenesis, maintenance of pluripotency, and metabolic flux. p53 inhibits gene expression by both direct and indirect means. Briefly, p53 can block transcription through direct DNA binding, association with transcription factors, and through the induction of genes whose functional products facilitate downstream repression. Indirect regulation of gene repression by p53 often involves induction of intermediary factors that fall into several categories: proteins (e.g. p21), microRNAs (e.g. miR-34a), and lincRNAs (lincRNA- p21). This dissertation discusses multiple aspects of p53-dependent gene repression and presents novel targets of p53-mediated regulation. Specifically, we have found that p53 down-regulates the transcription of the oncogenic transcription factor FoxM1. Mechanistically, this repression is largely dependent upon the p53-inducible gene p21, and consequently involves the Rb-family of tumor suppressors. Functionally, p53- dependent repression of FoxM1 contributes to the maintenance of a stable G2 cell cycle arrest in response to DNA-damage. In addition, we have identified PVT1 as a novel target of p53-transactivation. PVT1 encodes both spliced non-coding RNAs (ncRNA), as well as a series of microRNAs (miR-1204, miR-1205, miR-1206, miR-1207-5p, miR- 1207-3p and miR-1208). p53 upregulates PVT1 ncRNA, primary microRNAs, and mature miR-1204. Ectopic expression of miR-1204 induces changes in cell fate that are consistent with the role of p53 (cell death, cell cycle arrest), thus miR-1204 is likely to represent a functional target of p53 at the PVT1 locus. TABLE OF CONTENTS CHAPTER 1: INTRODUCTION.................................................................................... 1 DISCOVERY AND HISTORICAL PERSPECTIVE ........................................................................ 2 p53 AND CANCER ............................................................................................................... 3 p53: THE BASIC FACTS ....................................................................................................... 6 p53 THE TRANSCRIPTION FACTOR ....................................................................................... 7 TRANSCRIPTIONAL ACTIVATION .......................................................................................... 8 TRANSCRIPTIONAL REPRESSION........................................................................................ 10 Direct Transcriptional Repression mediated by p53 ................................................. 12 Direct/Indirect Repression ......................................................................................... 16 Indirect Repression .................................................................................................... 17 Indirect repression: p21/CDKN1A............................................................................. 19 Indirect repression: Non-coding RNA........................................................................ 21 microRNAs ................................................................................................................. 22 Long non-coding RNAs .............................................................................................. 25 p53 FUNCTION................................................................................................................. 27 Lessons from mouse models ....................................................................................... 27 p53 TARGET GENES: THE ROLE OF TRANSCRIPTIONAL ACTIVATION AND REPRESSION ........... 32 Apoptosis .................................................................................................................... 32 Apoptosis: A role for transcriptional repression ....................................................... 34 Cell cycle arrest/Senescence ...................................................................................... 36 G1/S checkpoint: p53 ................................................................................................. 37 G2/M Checkpoint: p53............................................................................................... 38 i Senescence: p53 ......................................................................................................... 42 Cell cycle arrest and senescence: The role of p53-mediated repression................... 46 Stem Cells and cancer stem cells: p53....................................................................... 49 Other tumor associated phenotypes: p53................................................................... 51 FOXM1 ........................................................................................................................... 52 REFERENCES ................................................................................................................... 58 CHAPTER 2: PRO-PROLIFERATIVE FOXM1 IS A TARGET OF P53- MEDIATED REPRESSION.......................................................................................... 90 ABSTRACT........................................................................................................................ 91 INTRODUCTION................................................................................................................ 91 RESULTS.......................................................................................................................... 92 DISCUSSION..................................................................................................................... 97 MATERIALS AND METHODS............................................................................................... 98 ACKNOWLEDGEMENTS ..................................................................................................... 99 REFERENCES ................................................................................................................... 99 CHAPTER 3: P53-DEPENDENT INDUCTION OF PVT1 AND MIR-1204.......... 102 ABSTRACT...................................................................................................................... 104 INTRODUCTION.............................................................................................................. 105 RESULTS........................................................................................................................ 111 DISCUSSION................................................................................................................... 118 MATERIALS AND METHODS............................................................................................. 123 ii ACKNOWLEDGEMENTS ................................................................................................... 128 REFERENCES ................................................................................................................. 129 FIGURE LEGENDS........................................................................................................... 136 FIGURES........................................................................................................................ 139 CHAPTER 4: PERSPECTIVES AND FUTURE DIRECTIONS............................ 145 PERSPECTIVES AND FUTURE DIRECTIONS......................................................................... 146 REFERENCES ................................................................................................................. 165 FIGURE LEGENDS........................................................................................................... 171 FIGURES........................................................................................................................ 174 iii LIST OF FIGURES 1.1 p53 represses multiple FoxM1-inducible targets……………………………………57 2.1 FoxM1 mRNA is downregulated in a p53-dependent manner……………………...92 2.2 The repression of FoxM1 is p21-dependent………………………………………...93 2.3 Retinoblastoma (Rb) family members have a role in FoxM1 mRNA repression……………………....................................................................................94 2.4 Nutlin is a potent FoxM1 repressor in two cancer cells lines which do not effectively repress FoxM1 in response to Daunorubicin…………………………....95 2.5 The p53-mediated repression of FoxM1 has a role in stable G2 arrest………….….96 3.1 p53 stimulates transcription at the PVT1 locus……………………………………139 3.2 Endogenous p53 binds to a canonical response element within the PVT1 locus….140 3.3 p53 directly binds the PVT1 RE in a sequence specific manner…………………..141 3.4 p53 facilitates transcriptional activity from the PVT1 RE………………………...142 3.5 p53 induces primary and mature miR-1204……………………………………….143 3.6 Ectopic miR-1204 the induces apoptosis or cell cycle arrest……….......................144
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