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UNIVERSITY of CALIFORNIA RIVERSIDE Investigations Into The UNIVERSITY OF CALIFORNIA RIVERSIDE Investigations into the Role of TAF1-mediated Phosphorylation in Gene Regulation A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Cell, Molecular and Developmental Biology by Brian James Gadd December 2012 Dissertation Committee: Dr. Xuan Liu, Chairperson Dr. Frank Sauer Dr. Frances M. Sladek Copyright by Brian James Gadd 2012 The Dissertation of Brian James Gadd is approved Committee Chairperson University of California, Riverside Acknowledgments I am thankful to Dr. Liu for her patience and support over the last eight years. I am deeply indebted to my committee members, Dr. Frank Sauer and Dr. Frances Sladek for the insightful comments on my research and this dissertation. Thanks goes out to CMDB, especially Dr. Bachant, Dr. Springer and Kathy Redd for their support. Thanks to all the members of the Liu lab both past and present. A very special thanks to the members of the Sauer lab, including Silvia, Stephane, David, Matt, Stephen, Ninuo, Toby, Josh, Alice, Alex and Flora. You have made all the years here fly by and made them so enjoyable. From the Sladek lab I want to thank Eugene, John, Linh and Karthi. Special thanks go out to all the friends I’ve made over the years here. Chris, Amber, Stephane and David, thank you so much for feeding me, encouraging me and keeping me sane. Thanks to the brothers for all your encouragement and prayers. To any I haven’t mentioned by name, I promise I haven’t forgotten all you’ve done for me during my graduate years. iv Dedication To my parents and my family for their unwavering support. You’ll never know how much its meant. v ABSTRACT OF THE DISSERTATION Investigations into the Role of TAF1-mediated Phosphorylation in Gene Regulation by Brian James Gadd Doctor of Philosophy, Graduate Program in Cell, Molecular and Developmental Biology University of California, Riverside, December 2012 Xuan Liu, Chairperson In response to cellular stresses, the transcription factor p53, a tumor suppressor, con- trols cell cycle arrest, DNA repair, or senescence by modulating the expression of target genes. Phosphorylation of p53 at threonine-residue 55 (p53-Thr55) by the TBP-associated factor 1 (TAF1), a subunit of the general transcription factor TFIID, suppresses the interac- tion of p53 with target genes and promotes nuclear export of p53, resulting in degradation of p53 and G1 cell cycle progression. TAF1-mediated p53-Thr55 phosphorylation in re- sponse to UV damage or high glucose (HG) treatment terminates the transcription of p21, an important regulator of G1 progression. In Chapter 2, as a first step to identify additional p53 target genes regulated by TAF1- mediated p53-Thr55 phosphorylation through genome-wide transcription and protein-DNA interaction assays, I recapitulated the HG-induced, TAF1-, and p53-dependent repression of p21 and confirmed the conditions under which maximal repression of p21 can be ob- served. Furthermore, my preliminary results suggest that HG can alter p53 protein levels in response to UV damage and provide a model for how HG increases the cancer risk of diabetics. vi In Chapter 3, I collected lists of p53- and TAF1- target genes, and HG-regulated genes via bioinformatics. I identified a set of genes commonly activated in response to different stress conditions: 12 p53-activated and 11 p53-repressed TAF1 target genes differentially expressed by HG, suggesting that these genes are regulated via TAF1-mediated p53-Thr55 phosphorylation. Furthermore, I describe a number of transcription factors representing putative targets for regulation via TAF-mediated phosphorylation. In Chapter 4, I describe tools to further investigate the relationship between TAF1 and p53. A 2x3’BS p21-LUC construct shall allow discernment of the role of different p53 DNA binding sites in the differential recruitment of TAF1 by DNA bound p53. Two TAF1 kinase mutant expression constructs shall facilitate the future elucidation of the TAF1 ki- nase domain responsible for p53-Thr55 phosphorylation in vivo. Finally, my preliminary results suggest that the p53 target gene NOXA is regulated via TAF1-mediated p53-Thr55 phosphorylation in response to UV, indicating that TAF1-mediated phosphorylation of p53- Thr55 may be involved in regulating the expression of a subset of p53 target genes. vii Contents 1 Introduction 1 1.1 Tumor Suppressor p53 . .1 1.2 TAF1 . 31 1.3 The TAF1/p53 Interplay . 38 2 High Glucose Induces TAF1-dependent Downregulation of p53 Protein Levels and Activity 77 2.1 Introduction . 77 2.2 Materials and methods . 81 2.3 Results . 84 2.4 Discussion . 90 2.5 Figures and Tables . 98 3 Bioinformatic Identification of Putative High glucose-induced TAF1 Regula- tory Targets 110 3.1 Introduction . 110 viii 3.2 Materials and Methods . 113 3.3 Results . 125 3.4 Discussion . 143 3.5 Figures and Tables . 158 4 Tools for Analysis of TAF1 and p53 197 4.1 Introduction . 197 4.2 Materials and Methods . 202 4.3 Results . 204 4.4 Discussion . 211 4.5 Figures and Tables . 218 5 234 5.1 Future Work . 242 5.2 Figures and Tables . 246 Appendix A 255 Appendix B 257 Appendix C 297 ix List of Figures 1.1 Overview of p53 Pathways . .2 1.2 Structure of the p53 protein . .4 1.3 Posttranslational Modification of the p53 protein . 24 1.4 TAF1 Structure . 35 2.1 A Working Model for p21 Downregulation by HG . 99 2.2 HG Downregulates p21 Expression . 101 2.3 HG-mediated Downregulation of p21 is p53-dependent . 102 2.4 HG-mediated Downregulation of p21 is TAF1-dependent. 103 2.5 HG Alters p53 Stability in UV-treated Cells. 104 3.1 High Glucose Regulatory Model . 159 3.2 Overview of Bioinformatic Analysis . 161 3.3 UV and IR responsive p53-dependent Genes Bound by p53 . 164 3.4 Comparison of p53 Target Genes Experimental and Literature Lists . 165 3.5 Venn Diagram Comparison of Stress-induced p53 Target Genes . 166 3.6 KEGG p53 Signaling Pathway Genes . 174 x 3.7 A Subset of p53 Target Genes are TAF1 Targets Regulated by HG. 175 3.8 p53, TAF1 and HG Regulated Genes . 177 3.9 A subset of TAF1-bound p53 Target Genes are Inversely Regulated by HG . 178 3.10 GO Analysis of p53-independent, HG-regulated TAF1 Target Genes . 179 3.11 KEGG Analysis of p53-independent, TAF1 Target Genes Regulated by HG 180 3.12 Enriched TFBS at p53-independent, TAF1 Targets Up- or Downregulated by High Glucose . 181 3.13 MHC Class I Genes are TAF1 Targets Downregulated by HG . 185 4.1 p21 Promoter and p53-DBS . 219 4.2 2x3’BS p21-LUC Map . 220 4.3 Construction of a p21LUC Construct Containing Two 3’ p53 DNA Binding Sites . 221 4.4 p53-driven Luciferase Expression from WT and 2x3’BS p21 Promoters . 222 4.5 Schematic Diagram of WT and Mutant TAF1 Proteins . 223 4.6 pcDNA3.1(∗)Map .............................. 224 4.7 pcDNA3.1(∗)FL TAF1 A2/N7 Ala Map . 225 4.8 pCMV-HAhTAF1 N1646Map . 226 4.9 Overexpression of TAF1 Constructs . 227 4.10 p21 Expression Following UV Damage . 228 4.11 NOXA May Be Regulated Similarly to p21 Following UV Damage . 229 5.1 TAF1 Regulation of p53 . 247 5.2 A Model for TAF1 Kinase Activity in Cell Cycle Progression . 248 xi C.1 p21-LUC 2x3’BS Construction Primers . 298 C.2 Primer Alignment . 302 xii List of Tables 2.1 Glucose Treatment Conditions . 100 3.1 Summary of Studies Used . 160 3.2 Direct p53 Target Gene List Sources . 162 3.3 p53 Binding Site Annotation . 163 3.4 TAF1 Target Genes: Promoter-bound TAF1 Gene Annotation . 169 3.5 HG Microarray Analysis Comparison . 170 3.6 HG-Induced Expression of Factors Related to the Working Model . 171 3.7 Gene Ontology Analysis for HG-Regulated Genes . 172 3.8 KEGG Analysis of HG Differentially Expressed Genes . 173 3.9 Transcription Factor Binding Sites Enriched at HG-downregulated, p53- independent, TAF1 Target Genes . 182 A.1 RT-PCR Primers . 256 B.1 p53 Target Genes Literature List . 258 B.2 p53 Target Genes Experimental List . 268 B.3 p53-Independent, HG Downregulated, TAF1 Bound GO Analysis . 294 xiii C.1 ChIP primers . 299 C.2 RT-PCR and RT-qPCR Primers . 300 xiv Chapter 1 Introduction 1.1 Tumor Suppressor p53 The p53 protein is an important tumor suppressor protein. At the molecular level, p53 functions as a transcription factor that can activate and repress target gene expression. p53 is present at low levels in healthy cells. In response to cell stress such as DNA damage the transcriptional activity and level of p53 are increased through mechanisms involving post- translational modification and protein stabilization, respectively. Active p53 1 can initiate a proper response to cellular stress: transient cell cycle arrest and DNA repair following moderate stress or permanent cell cycle arrest (senescence) or pre-programmed cell death (apoptosis) in cases of more severe insult. Activation of p53 occurs in response to a va- riety of cell stresses including DNA damage, hypoxia, nutrient deprivation, mitochondrial biogenesis stress, ribosomal biogenesis, spindle poisons, heat or cold shock, protein un- folding, and oncogene activation (Figure 1.1) [164]. In addition to DNA repair, cell cycle 1 1.1. TUMOR SUPPRESSOR P53 CHAPTER 1. INTRODUCTION and apoptosis, p53 also participates in auto-regulation, development, differentiation, an- giogenesis, migration, and metabolism [173, 214, 260]. Thus, the p53 protein serves as a central sensor of a variety of cell stresses in order to ultimately influence the integrity of the genome and the ability of the cell to divide.
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