Investigations on the roles of ubiquitin in the regulation of heat shock gene HSP70B 2

Acknowledgements I would like to thank the following people for making this thesis possible. My supervisor Assistant Professor Norbert Lehming for his invaluable insight and guidance. My mentor Miss Tan Yee Sun for her patience, concern and instruction. The staff in the lab, past and present for their professionalism, constant advice and support; Madams Chew Lai Ming, Cecilia Yang Caixin, Misses Siew Wee Leng, Nguyen Khahn Linh. Tay Ywee Chieh, Lim Mei Kee and Mister Leo Lim Mun Kuan. My fellow graduate students for all the camaraderie, aid and admittedly a certain degree of shared schandenfreude: Doctors Chew Boon Shang, He Hong Peng, Xue Xiaowei, Misses Zhao Jin, Vivian Tang Hui Ming, Rashmi Triparthi, Linda Lee Shu Yi and Mister Keven Ang KueLoong All the undergraduate students who have passed through the lab’s doors for their friendship and helping me understand that it is by teaching that we truly learn. Madams Wong Seong Leng, Rachel Anne Therese Teo Shi Hui, Misses Yu Jia, Chan Yu Mun, Karen Naduas, Celeste Lau, Nur Sabrina bte Sapari, Lim Hui Kheng, Wu Mei Hui, Agnes Chia Yi Fang, Yeo Jia Hui, Nafiza bte Ahmad, Misters Benjamin Xiao Junwen Daniel Wu Zheng’An, Sun Weiqi, Edwin Ang Lee Keong and Elvin Koh Wei Chuan. Special mention to Jason Goo Kian Sim for being the de facto “big brother” to all the other graduate students. Also to all the great people in the other labs on MD4 level that I had the privilege to work with. It would have been a much harder road without you all. i Table of Contents Title Page Chapter 1: Introduction 1.1: The Process and Mechanisms of Transcription 1.1.1 Introduction to Transcription and the Transcriptional Machinery 1.1.2 The Eukaryotic Transcriptional Machinery 1.2 The Mediator Complex 1.2.1 Introduction to the Mediator Complex 1.2.2 Structure and Function of the Mediator Complex 1.3 The Ubiquitin Proteosome System 10 1.3.1 The Discovery of the Ubiquitin Proteomsome System 10 1.3.2 The Structure and Function of the 26S Proteosome 13 1.3.3 The Machinery and Process of Ubiquitination in Brief 16 1.3.4 E4 Ubiquitin Ligases in the Elongation of Polyubiquitin Chains 19 1.3.5 Ubiquitin-Proteosome Adaptors 23 1.4 The Non-proteolytic Roles of Ubiquitin 24 ii 1.4.1 An Introduction to the Role of Ubiquitin as a Signaling Molecule 24 1.4.2 Ubiquitin as a Signaling Molecule in the Regulation of Transcription Factors 24 1.4.3 An Introduction to Mono-ubiquitination as a Signaling Molecule 26 1.4.4 Ubiquitin as a Mediator of Protein Interaction 28 1.4.5 Ubiquitin in the Regulation of Endocytosis and Intercellular Trafficking 29 1.4.6 Ubiquitin’s Role in DNA Repair 33 1.4.7 Signaling by Ubiquitin-like Molecules 34 1.5 The Role of Mediator, Ubiquitin and the Proteosome in Transcriptional Regulation 38 1.5.1 Introduction to Transcriptional Regulation in Eukaryotes 38 1.5.2 Mediator Complex in Transcriptional Regulation 39 1.5.3 The Role of Ubiquitin in Transcriptional Regulation 40 1.5.4 The Proteolytic Role of the Proteosome in the Regulation of Transcription 42 1.5.4 The Non-Proteolytic Role of the Proteosome in the Regulation of Transcription 47 1.5.6 The Role of Ubiquitin and the Proteosome System Acting in Unison in Transcriptional Regulation 52 1.5.7 Common Components of the Transcriptional Machinery and the Ubiquitin Proteosome System 55 iii 1.6 Theoretical Basis and Systemic Objectives of This Study 57 Chapter 2: Materials and Methods 58 2.1 Molecular Tools for the Expression of Proteins 58 2.1.1 Cloning of Tagged Constructs into Vectors 58 2.1.2 Cloning Stategies 59 2.1.3 Polymerase Chain Reaction (PCR) 59 2.1.4 Cloning of Gene into Vectors 60 2.1.5 Transformation and Inoculation 60 2.1.6 Low-yield Purification of Plasmid DNA (Miniprep) 61 2.1.7 Qualification of Miniprep product 62 2.1.8 DNA Sequencing 62 2.2 Synthesis of Constructs for RNA Interference of hSKP1 and hSRB7/MED21 63 2.2.1 Design of siRNA pSuper Constructs 63 2.2.2 Annealing of Constructs 63 2.3 Transfection of High Quality Plasmids into HeLa Cells 64 iv 2.3.1 Retransformation 65 2.3.2 Medium-yield Purification of Plasmid DNA (Midi-prep) 65 2.3.3 Transfection of Plasmids into Human Cells 67 2.4 Tools for the Identification, Purification and Visualization of Proteins 70 2.4.1 Purification of GST and MYC-tagged constructs 70 2.4.2 Coimmunoprecipitation of Mediator and SCF Components 71 2.4.3 Western Blot 72 2.4.4 Stripping Western Blots 76 2.4.5 Split-ubiquitin Screens 76 2.5 Tools for mRNA Extraction and Quantification 78 2.5.1 Transfection of siRNA constructs and Treatments Prior to mRNA Isolation 78 2.5.2 Isolation of mRNA 78 2.5.3 DNAaseI Treatment and Reverse Transcription 80 2.5.4 Real-Time Quantative PCR 81 2.6 Chromatin Immunoprecipitation 83 v Chapter 3: Results 86 3.1 Introduction to Skp1 and Relevant Molecular Tools 86 3.2 HSP70B’ as an Inducible Gene for Study 92 3.3 The Effects of Skp1 Depletion on HSP70B’ Expression 96 3.4 Proteosomal Inhibition of HSP70B’ and its Interaction With siSkp1 99 3.5 Srb7 as an Interacting Partner of Skp1 104 3.6 The Role of Med21/hSrb7 in the Regulation of HSP70B’ 109 3.7 The Effect of the RNA Interference of Med21/hSrb7 and its Effects on Proteosomal Inhibition of HSP70B’ Induction 113 3.8 The Interaction between Skp1 and Mediator Components 118 3.9 The Interaction between Med6 and Cul1 121 3.10 Molecular Tools Concerning Hsf1 130 3.11 The Effect of siHSF1 on HSP70B’ Induction 133 3.12 Determining the Existence of Ubiquitinated Species of Hsf1 137 3.13 The Stability of Hsf1 141 3.14 The Presence of Hsf1 at the HSP70B’ Promoter 149 vi Chapter 4: Discussion 158 4.1 Key Findings of the Project 158 4.2 The Regulation of HSP70B’ and its Relevance to Human Health and Disease 159 4.3 Proteosome Inhibitors and Hsp70B’ 164 4.4 The Regulation of HSP70B’ by Hsf1 167 4.5 The Role of Hsf1 in Human Health and Disease 173 4.6 Conclusion 176 Bibliography 177 References 177 Websites 197 Appendix 198 Common Stock Solutions 198 Publications 198 vii Summary Transcription is one of the most fundamental processes in a living cell and as such, it is no surprise that it is tightly regulated on many levels. The post-translational modification of transcription factors is one means by which the cell achieves transcriptional regulation. Of particular interest to us is the ubiquitination of transcription factors. Ubiquitin traditionally serves as a proteolytic signal in the form of K48 polyubiquitin chains that are recognized by the 26S proteosome. However it is now accepted that mono-ubiquitin as well as K63 polyubiquitin chains are both signaling motifs outside of the context of proteosomal degradation (Hicke et al., 2005). Each of these modifications has their role in the regulation of transcription. In this project we investigated the role of ubiquitin in the regulation of the heat shock gene HSP70B’. Through RNA interference of SKP1, a component of an ubiquitin ligase (Zhou, et al., 1998), we demonstrated that the loss of ubiquitination can severely reduce the induction of HSP70B’ by heat shock. However this effect is not necessarily the result of a loss of proteosomal degradation affecting a transcription factor. The application of a proteosome inhibitor induces the expression of HSP70B’ and RNA interference of SKP1 likewise can reduce this increase in expression. This would imply that ubiquitination signaling, rather than proteosomal degradation, is a key factor in the regulation of HSP70B’. Split-ubiquitin screens performed to discover if Skp1 has interacting partners outside of its native ubiquitin ligase complex found that Mediator component Srb7 interacts with Skp1 in vitro. In viii vivo evidence for this interaction was found in a co-immunoprecipitation where human Skp1 could immunoprecipitate Med21, the human homologue of Srb7, and vice versa. RNA interference of MED21 could reduce the induction of HSP70B’ by heat shock or proteosomal inhibition much in the same manner as RNA interference targeted at SKP1 did. This implies a functional relationship between these proteins in the regulation of HSP70B’. Coimmunoprecipitation experiments showed that components of the SCF (Skp1-Cullin-F-box protein) complex that Skp1 belongs to can immunoprecipitate components of the Mediator Complex and that the reverse is also true. This could indicate that these two complexes have a hereinto uncharacterized relationship in the regulation of transcription. Subsequent investigations found that the heat shock transcription factor Hsf1 is a regulator of HSP70B’ expression; RNA interference targeted at HSF1 causes a strong reduction in HSP70B’ induction that can be ameliorated by the application of proteosome inhibitor. Coimmunoprecipitation experiments demonstrated that Hsf1 has abundant ubiquitinated species in vivo. Overall our data favors a model where ubiquitination is a moiety that activates the transcription factor Hsf1 to drive HSP70B’ expression. Over time, this ubiquitinated Hsf1 is polyubiquitinated and degraded, limiting its active lifespan which accounts for the activity of both proteosome inhibitors and siRNA targeted at SKP1. Our findings have great relevance to human health and disease; the regulation of heat shock proteins and Hsf1 implicated in various diseases (Khaleque et al., 2005) while a proteosome inhibitor is used in chemotheraphy (San Miguel et al., 2008). (500 words) ix List of Tables Table Page Table 1: DNA and LipofectANIME Ratios for Transfection 69 Table 2: Western Blot Primary and Secondary Antibodies 75 Table 3: Primers Used for ChIP 85, 152 List of Figures Figure Page Figure 1: Immunoprecipitation of Skp1 with Endogenous Antibody and Protein G Sepharose Beads 87 Figure 2: MYC-hSkp1 is Expressed in the Cell 88 Figure 3: Immunoprecipitation of Skp1 with Endogenous Antibody and Protein G Sepharose Beads 89 Figure 4: Knockdown of Skp1 by a pSuper construct 90 Figure 4.1: Quantification of “Knockdown of Skp1 by a pSuper construct” 91 Figure 5: Measurement of HSP90 and HSP70B’ Heat Induction 94 Figure 6: siRNA Treatment Can Reduce the Expression of SKP1 95 Figure 7: siSKP1 Treatment Reduces HSP70B’ Induction Upon Heat-shock 96 x Figure 8: hSkp1 siRNA Can Affect the Induction of HSP70B’ By MG132 100 Figure 9: MG132 Treatment Induces HSP70B’ In Addition to Heat-shock 102 Figure 10 hSkp1 siRNA Can Affect the Induction of HSP70B’ 103 Figure 11: A Split-Ubiquitin Screen identified hSkp1p as a hSrb7p-interacting Protein 106 Figure 12: hSkp1 Interacts with hSrb7 in vitro 108 Figure 13: siRNA Treatments Can Reduce the Expression of MED21 110 Figure 14: siMED21 Treatment Reduces HSP70B’ Induction Upon Heat-shock 111 Figure 15: GAPDH Ct Values Reflect No General Transcription Defect in the Cell 112 Figure 16: siMED21 Can Affect the Induction of HSP70B’ By MG132 113 Figure 17: siMED21 Can Affect the Induction of HSP70B’ 114 Figure 18: Co-immunoprecipitation of Med6 by MYC-Skp1 using MYC-tagged Beads 120 Figure 19: Immunoprecipitation of hCul1 122 Figure 20: Immunoprecipitation of hMed6 123 Figure 21: Co-immunoprecipitation of Cul1 by Med6 with Protein A and Protein G 124 Figure 22: Co-immunoprecipitation of Med6 by Cul1 with Protein A and Protein G 125 Figure 23: siRNA Treatment Has Only Modest Effect on Hsf1 Expression 130 xi Figure 23.1: Quantification of “siRNA Treatment Has Only Modest Effect on Hsf1 131 Expression” Figure 24: The Expression of HSF1 Can Be Reduced by siRNA Treatment 132 Figure 25 siHSF1 Can Lower the Induction of HSP70B’ After Proteosome Inhibition 133 Figure 26: siHSF1 Can Lower the Induction of HSP70B’ After Heat Shock 134 Figure 27: Immunoprecipitation of ubiquitinated Hsf1 with α-Hsf1 and Protein A/G 138 Figure 28: Immunoprecipitation of ubiquitinated Hsf1 by α-Ub and Protein G 139 Figure 29: ChIP Targeting the HSP70B’ Promoter using MYC-Skp1 140 Figure 30: Hsf1 is Stable in a 37°C Cycloheximide Chase 142 Figure 30.1: Cul1 as a Loading Control for Hsf1 Stability 142 Figure 30.2: Quantification of “Hsf1 is Stable in a 37°C Cycloheximide Chase” 143 Figure 31: Hsf1 is Stable in a 37°C Cycloheximide Chase 144 Figure 32: Excessive Heat Shock Leads to Degradation of Hsf1 146 Figure 32.1: Cul1 as a Loading Control for Hsf1 Stability 146 Figure 32.2: Quantification of “Excessive Heat Shock Leads to Degradation of Hsf1” 147 Figure 33:Crosslinking Leads to Loss of Protein Signal 150 Figure 34: HSP70B’ Promoter 152 xii Figure 35: ChIP of Ubiquitinated Hsf1 Shows No Promoter Occupancy at HSP70B’ Promoter 153 Figure 36: ChIP Sample DNA Concentration (ng/µL) 157 List of Illustrations Illustration Page Illustration 1: Transcription Preinitiation Complex Assembly Illustration 2: Ubiquitin Proteosome System 17 Illustration 3: Split Ubiquitin System 77 Illustration 4: Hypothetical Model of HSP70B’ Regulation 171 List of Symbols None List of Conventions Gene name with only first letter capitalized, i.e. Protein Hsf1 Gene name entirely capitalized, i.e. HSF1 Protein; often an abbreviation, complex or compound name xiii Gene name entirely capitalized and italicized, i.e. HSF1 Gene or mRNA α-[protein name] Antibody specific for the named protein si[gene name] Small interfering RNA construct specific for the named gene’s mRNA h[protein name or gene name] Refers to a human protein or gene; used to distinguish between Saccharomyces cerevisiae and Homo Sapiens proteins or genes when both are relevant to the discussion at hand List of Common Abbreviations All abbreviations used are explained in the text but this table contains some of the most commonly used abbreviations for ease of reference. APIS The 19S ATPase proteins independent of 20S Complex ATP Adenosine Triphosphate CHIP C-terminus of Hsc70-interacting protein ChIP Chromatin Immunoprecipitation CIITA Class II Transactivator xiv CTD C-terminal Domain DNA Deoxyribonucleic Acid DUB Deubiquitinating Enzyme GAPDH Glyceraldehyde-3-phosphate Dehydrogenase GST Glutathione-S-Transferase HA Haemagglutinin HAT Histone Acetyltransferase HDAC Histone Deacetylase HRP Horseradish Peroxidase HSE Heat Shock Element MHCII Major Histocompatability Class II mRNA Messenger Ribonucleic Acid MYC Epitope derived from the c-MYC gene xv PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction RNA Ribonucleic Acid RT qPCR Reverse Transcription-coupled Quantative Real-time PCR SCF Complex Skp1 - Cullin - F-box Protein Complex siRNA Short interfering Ribonucleic Acid Ub Ubiquitin UBD Ubiquitin-binding Domain UBP Ubiquitin Protease UPS Ubiquitin Proteosome System USP Ubiquitin Specific Protease xvi [...]... siRNA Treatment 1 32 Figure 25 siHSF1 Can Lower the Induction of HSP70B After Proteosome Inhibition 133 Figure 26 : siHSF1 Can Lower the Induction of HSP70B After Heat Shock 134 Figure 27 : Immunoprecipitation of ubiquitinated Hsf1 with α-Hsf1 and Protein A/G 138 Figure 28 : Immunoprecipitation of ubiquitinated Hsf1 by α-Ub and Protein G 139 Figure 29 : ChIP Targeting the HSP70B Promoter using MYC-Skp1 140... Treatment Reduces HSP70B Induction Upon Heat- shock 111 Figure 15: GAPDH Ct Values Reflect No General Transcription Defect in the Cell 1 12 Figure 16: siMED21 Can Affect the Induction of HSP70B By MG1 32 113 Figure 17: siMED21 Can Affect the Induction of HSP70B 114 Figure 18: Co-immunoprecipitation of Med6 by MYC-Skp1 using MYC-tagged Beads 120 Figure 19: Immunoprecipitation of hCul1 122 Figure 20 : Immunoprecipitation... Affect the Induction of HSP70B By MG1 32 100 Figure 9: MG1 32 Treatment Induces HSP70B In Addition to Heat- shock 1 02 Figure 10 hSkp1 siRNA Can Affect the Induction of HSP70B 103 Figure 11: A Split -Ubiquitin Screen identified hSkp1p as a hSrb7p-interacting Protein 106 Figure 12: hSkp1 Interacts with hSrb7 in vitro 108 Figure 13: siRNA Treatments Can Reduce the Expression of MED21 110 Figure 14: siMED21... Immunoprecipitation of hMed6 123 Figure 21 : Co-immunoprecipitation of Cul1 by Med6 with Protein A and Protein G 124 Figure 22 : Co-immunoprecipitation of Med6 by Cul1 with Protein A and Protein G 125 Figure 23 : siRNA Treatment Has Only Modest Effect on Hsf1 Expression 130 xi Figure 23 .1: Quantification of “siRNA Treatment Has Only Modest Effect on Hsf1 131 Expression” Figure 24 : The Expression of HSF1 Can... explained in the text but this table contains some of the most commonly used abbreviations for ease of reference APIS The 19S ATPase proteins independent of 20 S Complex ATP Adenosine Triphosphate CHIP C-terminus of Hsc70-interacting protein ChIP Chromatin Immunoprecipitation CIITA Class II Transactivator xiv CTD C-terminal Domain DNA Deoxyribonucleic Acid DUB Deubiquitinating Enzyme GAPDH Glyceraldehyde-3-phosphate... 2: Ubiquitin Proteosome System 17 Illustration 3: Split Ubiquitin System 77 Illustration 4: Hypothetical Model of HSP70B Regulation 171 List of Symbols None List of Conventions Gene name with only first letter capitalized, i.e Protein Hsf1 Gene name entirely capitalized, i.e HSF1 Protein; often an abbreviation, complex or compound name xiii Gene name entirely capitalized and italicized, i.e HSF1 Gene. .. α-[protein name] Antibody specific for the named protein si [gene name] Small interfering RNA construct specific for the named gene s mRNA h[protein name or gene name] Refers to a human protein or gene; used to distinguish between Saccharomyces cerevisiae and Homo Sapiens proteins or genes when both are relevant to the discussion at hand List of Common Abbreviations All abbreviations used are explained in the. .. “Excessive Heat Shock Leads to Degradation of Hsf1” 147 Figure 33:Crosslinking Leads to Loss of Protein Signal 150 Figure 34: HSP70B Promoter 1 52 xii Figure 35: ChIP of Ubiquitinated Hsf1 Shows No Promoter Occupancy at HSP70B Promoter 153 Figure 36: ChIP Sample DNA Concentration (ng/µL) 157 List of Illustrations Illustration Page Illustration 1: Transcription Preinitiation Complex Assembly 3 Illustration 2: ... Stable in a 37°C Cycloheximide Chase 1 42 Figure 30.1: Cul1 as a Loading Control for Hsf1 Stability 1 42 Figure 30 .2: Quantification of “Hsf1 is Stable in a 37°C Cycloheximide Chase” 143 Figure 31: Hsf1 is Stable in a 37°C Cycloheximide Chase 144 Figure 32: Excessive Heat Shock Leads to Degradation of Hsf1 146 Figure 32. 1: Cul1 as a Loading Control for Hsf1 Stability 146 Figure 32. 2: Quantification of “Excessive... Reaction RNA Ribonucleic Acid RT qPCR Reverse Transcription-coupled Quantative Real-time PCR SCF Complex Skp1 - Cullin - F-box Protein Complex siRNA Short interfering Ribonucleic Acid Ub Ubiquitin UBD Ubiquitin- binding Domain UBP Ubiquitin Protease UPS Ubiquitin Proteosome System USP Ubiquitin Specific Protease xvi . in the regulation of transcription. In this project we investigated the role of ubiquitin in the regulation of the heat shock gene HSP70B . Through RNA interference of SKP1, a component of. Transcription 42 1.5.4 The Non-Proteolytic Role of the Proteosome in the Regulation of Transcription 47 1.5.6 The Role of Ubiquitin and the Proteosome System Acting in Unison in Transcriptional Regulation. 62 2. 1.8 DNA Sequencing 62 2. 2 Synthesis of Constructs for RNA Interference of hSKP1 and hSRB7/MED21 63 2. 2.1 Design of siRNA pSuper Constructs 63 2. 2 .2 Annealing of Constructs 63 2. 3