THE ROLE OF TRIP-BR PROTEINS IN THE REGULATION OF MAMMALIAN GENE TRANSCRIPTION AND CELL CYCLE PROGRESSION KHE GUAN, SIM (BSc & ARCS, Imperial College of Science, Technology and Medicine, University of London) A THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHICAL DOCTOR (PhD) INSTITUTE OF MOLECULAR AND CELL BIOLOGY (IMCB) AND DEPARTMENT OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2003 i ACKNOWLEDGEMENTS I wish to acknowledge the following kind individuals who made this work possible First of all, I thank Dr Stephen Hsu I-Hong (MD PhD), to whom I owe an immense debt of gratitude for his support and encouragement, for his enlightening teachings and guidance, and for his invaluable mentorship and friendship He has created a unique and yet conducive environment within which good science is learnt and practiced and good spiritual values are nurtured and inculcated I am grateful to the members of my supervisory committee from IMCB, Willian Chia, Paramjeet Singh and Hans Uli-Bernard for constantly monitoring my progress and pointing me in the right research direction I also wish to thank the Medicine Faculty of NUS and IMCB for the special opportunity to undertake my research training under a cross-faculty collaborative program No words can express my appreciation to all the beloved members of the Laboratory of Molecular Nephrology and Gene Regulation, who made the lab such an enjoyable place to work and learn Special thanks to fellow colleagues Sharon Thio, Christopher Yang, Shahidah, Jit Kong and Chui Sun for their moral and technical supports, for reviewing my manuscripts and for many constructive and insightful discussions I also wish to acknowledge Olivia Chao, for her generous friendship and help in obtaining reagents for some key experiments My utmost gratitude to my teachers, Charlie and Linda Lee, and all the great members of the SOONYETM organization, for opening my eyes to the true meaning of ii the proverb “It’s your attitude, not your aptitude that will determine your altitude in life” To my beloved family – Mom and Khe Chai, endless thanks for your love and support Most importantly, I send all my love to my angel, my wife Ally, who made everything possible through her love and spiritual support, who gave me the strength and courage to carry on, and who gave me the reason to succeed Finally, I wish to convey my heartfelt gratitude to all those kind people whom I neglect to mention by name KG, SIM April, 2003 iii TABLE OF CONTENTS Page Title i Acknowledgements ii Table of Contents iv List of Figures & Tables x List of Abbreviations xvi Presentations & Publications arising from PhD Thesis xviii Abstract Introduction The Plant Homeodomain (PHD) zinc finger and the bromodomain 1.1 PHD zinc fingers and bromodomains are evolutionarily conserved secondary structural protein motifs 1.2 The PHD zinc fingers and the bromodomains: functional implications 1.2.1 Protein motifs with biological significance 1.2.2 The role of PHD zinc fingers and the bromodomains 10 in the regulation of eukaryotic gene transcription Transcriptional regulators interacting with the PHD zinc finger 15 and/or the bromodomain (TRIP-Br) 2.1 Historical perspective 15 2.2 The structural features of the TRIP-Br proteins 17 2.3 The functional properties of the TRIP-Br proteins 18 2.3.1 The unique ability to interact with the PHD zinc 18 finger and/or the bromodomain iv 2.3.2 The TRIP-Br proteins possess potent acidic 19 transactivation domains 2.3.3 Co-regulation of the E2F-1/DP-1 transcriptional 19 activity 2.3.4 Functional significance of the PHD-bromodomain- 20 interacting potentials of the TRIP-Br proteins 2.4 The TRIP-Br proteins: a novel class of cell cycle regulator 21 2.4.1 The mammalian cell cycle 21 2.4.2 Functional relationships between the TRIP-Br 27 proteins and the E2F family of transcription factors 2.4.3 Functional interactions between the TRIP-Br 30 proteins and the cell cycle regulatory protein, cyclin A 2.4.4 Cell cycle regulated expression of hTRIP-Br1 31 2.4.5 hTRIP-Br1, a Cdk4-interacting regulatory protein 31 2.4.6 The integrator model of TRIP-Br protein function in 32 cell cycle regulation Materials and Methods Materials 1.1 Plasmid DNA and cDNA clones 34 1.2 Biochemical reagents 34 1.3 Synthetic peptides 35 1.4 Synthetic oligonucleotides 35 1.5 Bacterial strains 36 1.6 Tissue culture cell lines 36 2.1 Maintenance and Handling of Tissue Culture Cells 37 2.2 Preparation of Competent Bacterial Cells 37 2.3 Transformation and Maintenance of Plasmid DNA 38 Methods v Clones 2.4 Mini- and Maxi-scale Preparation of Plasmid DNA 38 from Bacteria 2.5 Quantitation and Purity Assessment of DNA 39 2.6 Screening of transformants for positive clones 39 2.7 Gel electrophoresis of DNA products 39 2.8 Preparation of Protein by in vitro Translation 39 2.9 Preparation of Peptide Stock Solutions 40 2.10 Preparation of Proteins from Tissue Culture 40 2.11 Analysis of Proteins by Polyacrylamide Gel 40 Electrophoresis (PAGE) 2.12 Western Blot for Protein Detection 41 2.13 Electro-mobility Shift Assays (EMSA) 41 2.14 Treatment of Cells with Decoy Peptides 42 2.15 Confocal Microscopy 42 2.16 Measurement of Peptide Internalization Efficiency 43 by Fluorescence-activated Cell Sorting (FACS) 2.17 DNA Transfection and Sequential β- 43 Galactosidase/Luciferase Assays 2.18 DNA Enzyme Transfection 44 2.19 Semi-quantitative Reverse Transcription coupled to 44 Polymerase Chain Reaction (RT-PCR) 2.20 Cell Proliferation Assays 46 2.21 Determination of Cell Number 47 2.22 Colony Formation Assay 48 2.23 Cell Cycle Profile and TUNEL Staining Analyses by 48 Flow Cytometry 2.24 Cell Synchronization at the G2/M Boundary and 49 Cell Cycle Progression Analyses 2.25 Caspase Assay 49 2.26 Protein Decay Analysis 50 vi 2.27 Immunoprecipitation Assay (IP) 50 Results Decoy peptides as molecular tools to probe the function of the PHD-bromodomain-interacting domain of TRIP-Br proteins 1.1 Designing peptides that antagonize physical 51 interactions between TRIP-Br proteins and PHD zinc fingers and/or bromodomain-containing proteins 1.2 Evaluation of decoy peptide-mediated blocking 55 activity in vitro 1.3 Evaluation of the cell-penetrating properties and 60 blocking activity of the TRIP-Br decoy peptides in vivo TRIP-Br decoy peptides reveal novel functions for TRIP-Br proteins in the regulation of E2F-dependent transcriptional activity 2.1 Decoy peptide treatment causes repression of an 64 artificial E2F-responsive reporter gene in vivo 2.2 The decoy peptides differentially down-regulate the 67 expression of endogenous E2F-responsive genes TRIP-Br decoy peptides impose a proliferative block 3.1 The TRIP-Br decoy peptides inhibit DNA synthesis 70 as assessed by BrdU incorporation assay 3.2 The TRIP-Br decoy peptides suppress cellular 71 proliferation vii The integrator function of the TRIP-Br proteins is implicated in the regulation of cyclin E expression during cell cycle progression 4.1 *Br1 or *Br2 perturbs the timing and the amplitude 77 of cyclin E protein expression 4.2 *Br1 or *Br2 does not alter the steady state level of 80 cyclin E mRNA transcript levels 4.3 80 Fbxw7 4.4 *Br1 or *Br2 down-regulates the expression of 84 Fbxw7 is a novel E2F-responsive and TRIP-Br coregulated gene Treatment with TRIP-Br decoy peptides is associated with subdiploidy 5.1 The TRIP-Br decoy peptides induce accumulation 89 of a sub-G1 population 5.2 Sub-diploidy induced by *Br1 Is biochemically 90 distinct from that triggered by *Br2 5.3 Sub-diploidy elicited by *Br1 or *Br2 is caspase- 95 independent 5.4 Incomplete DNA replication: an alternative 100 mechanism for sub-diploidization DNA enzymes targeting TRIP-Br mRNA inhibit serum-inducible fibroblast proliferation 6.1 E-Br1 or E-Br2 DNA enzymes specifically “knock 110 down” serum-induced hTRIP-Br1 or hTRIP-Br2 gene transcription 6.2 E-Br inhibits serum-induced WI-38 cell proliferation 116 6.3 E-Br DNA enzymes prevent serum-induced S 119 phase entry viii 6.4 E-Br DNA enzymes prevent serum-induced cyclin E 122 expression Discussions 128 References 142 ix LIST OF FIGURES & TABLES FIGURE 1: Schematic illustration of the PHD zinc finger and the bromodomain FIGURE 2: Conservation of the PHD zinc finger across evolutionary boundaries FIGURE 3: Putative domain structure of human TRIP-Br proteins 16 FIGURE 4: The mammalian cell cycle 22 FIGURE 5: The two possible mechanisms of pocket protein/E2F-DP complex-mediated repression of gene transcription in G1/G0 cells 26 FIGURE 6: Structural organization of mammalian E2F and DP proteins 28 FIGURE 7: The integrator model of TRIP-Br Protein Function 33 FIGURE 8: Schematic illustration of the decoy peptide antagonism strategy 52 FIGURE 9: Schematic representation of the TRIP-Br decoy peptides 54 FIGURE 10: Electro-mobility shift analysis (EMSA) demonstrating assembly of the DNA-GAL4/KRIP1/TRIP-Br super-shift complexes in vitro 57 x Being a newly identified E2F-responsive gene, the transcriptional activity of NPAT may be specifically co-regulated by the TRIP-Br2 integrator function in a manner analogous to dhfr and DNA Polymerase α Antagonism of this function using *Br2 is proposed to affect genomic stability and render genomic DNA more susceptible to non-caspase-mediated endonucleolytic degradation, with the consequential exposure of 3’-hydroxy DNA ends The TRIP-Br genes were found to be induced in response to serum stimulation (Figure 23A) To gain further insights into the role of the TRIP-Br proteins in seruminducible cell cycle progression, the TRIP-Br1 and TRIP-Br2 genes in WI-38 fibroblasts were specifically knocked-down using DNA enzymes targeting the TRIPBr1 or TRIP-Br2 mRNA transcripts Knocking-down TRIP-Br1 and TRIP-Br2 in WI-38 fibroblasts effectively suppressed serum-induced cellular proliferation, suggesting that the TRIP-Br proteins are physiologically involved in the execution of serum-induced cell cycle progression The anti-proliferative effects imposed by the DNA enzymes could be overridden through combined over-expression of E2F-1/DP1/cyclin E/Cdk2, indicating that the TRIP-Br proteins target one or more regulatory steps upstream of cyclin E/Cdk2 in the serum-inducible cell cycle signaling pathway These observations are consistent with the recent finding that hTRIP-Br1/p34SEI-1 plays an important role in augmenting the activation of cyclin D/Cdk4 kinase activity 49 Cyclin D-associated kinase activities play an integral role in serum-responsive intracellular signal transduction that contributes to the G0 to G1 transition during serum-induced cell cycle progression 38, 46 Thus, down-regulating TRIP-Br1 through the action of the E-Br1 DNA enzyme is expected to attenuate the kinase activity of cyclin D/Cdk4 in response to serum stimulation, thereby imposing a proliferative 138 block Based on the results presented in Figure 26D, TRIP-Br2 may be involved in CAK-mediated activation of Cdk4 E-Br2-mediated TRIP-Br2 knock-down is predicted to prevent full activation of Cdk4, thereby blocking cyclin E gene induction and S phase entry Measurements of Cdk4 and Cdk7 activity would lend further support to the notion that TRIP-Br2 is required for CAK-mediated activation of Cdk4 In summary, the decoy peptide antagonism and the DNA enzyme gene knock down approaches have unraveled some of the key physiological functions of the TRIP-Br proteins in the regulation of mammalian cell cycle The series of decoy peptide studies have provided physiological evidence in support of an important role for the PHD-bromodomain-interacting property (integrator function) of TRIP-Br1 and TRIP-Br2 in the regulation of the well-defined transcriptional and cell cycle regulatory pathway mediated by the E2F family of transcription factors (Figure 28) Through modulating the Fbxw7 gene activity, the TRIP-Br integrator function controls the cell cycle-dependent accumulation of cyclin E protein, which in turn coordinates the proper expression of Geminin The coordinated expression of Geminin, in concert with the regulation of dhfr, DNA Polα and cdc2, ensures proper initiation and execution of DNA replication as well as cell cycle phase-transitions The series of studies involving DNA enzymes has demonstrated the physiological involvement of both TRIP-Br1 and TRIP-Br2 in executing serum-inducible cellular proliferation Deficiency of either TRIP-Br family member results in a proliferative block, indicating that TRIP-Br1 and TRIP-Br2 have distinct roles in the regulation of serum-inducible cell cycle progression (Figure 29) 139 $ @ 8& &0 < & α &0 -0 =#- 0 / 80 - : * 8& # 0= & & 0 0 0 & 0 TRIP-Br integrator function in the regulation of E2F-dependent transcription and cell cycle progression 140 & 0 80 & / , & < &0 &0 09 &7 & &9 / & / & 0 , 0= / & 3& , , # 0= 0 , Regulation of serum-inducible cell cycle progression by TRIP-Br 141 REFERENCES Aasland, R., Gibson, T J., and Stewart, A F The PHD finger: implications for chromatin-mediated transcriptional 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and. .. Nevertheless, there are several lines of indirect but compelling evidence inferring a role for the TRIP- Br proteins in regulation of cell cycle progression (see 2.4.2 – 2.4.6): The TRIP- Br proteins regulate