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

iii 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

i

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 Kue-Loong

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 1 that I had the privilege to work with It would have been a much harder road without you all

ii

Table of Contents

1.1.1 Introduction to Transcription and the Transcriptional Machinery 1

1.2.2 Structure and Function of the Mediator Complex 7

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

iii

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

1.5 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

iv

1.6 Theoretical Basis and Systemic Objectives of This Study 57

2.1.6 Low-yield Purification of Plasmid DNA (Miniprep) 61

2.2 Synthesis of Constructs for RNA Interference of hSKP1 and hSRB7/MED21 63

2.3 Transfection of High Quality Plasmids into HeLa Cells 64

v

2.3.2 Medium-yield Purification of Plasmid DNA (Midi-prep) 65

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

2.5.1 Transfection of siRNA constructs and Treatments Prior to mRNA Isolation 78

vi

3.1 Introduction to Skp1 and Relevant Molecular Tools 86

3.3 The Effects of Skp1 Depletion on HSP70B’ Expression 96

3.4 Proteosomal Inhibition of HSP70B’ and its Interaction With siSkp1 99

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

3.12 Determining the Existence of Ubiquitinated Species of Hsf1 137

vii

4.2 The Regulation of HSP70B’ and its Relevance to Human Health and Disease 159

viii

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

ix

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’

Co-immunoprecipitation 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

Co-immunoprecipitation 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)

x

List of Tables

Table 1: DNA and LipofectANIME Ratios for Transfection 69

Table 2: Western Blot Primary and Secondary Antibodies 75

List of Figures

Figure 1: Immunoprecipitation of Skp1 with Endogenous Antibody and Protein G

Sepharose Beads

87

Figure 3: Immunoprecipitation of Skp1 with Endogenous Antibody and Protein G

Sepharose Beads

89

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

xi

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 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 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

xii

Figure 23.1: Quantification of “siRNA Treatment Has Only Modest Effect on Hsf1

Expression”

131

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

xiii

Figure 35: ChIP of Ubiquitinated Hsf1 Shows No Promoter Occupancy at HSP70B’

Promoter

153

List of Illustrations

Illustration 1: Transcription Preinitiation Complex Assembly 3

Illustration 4: Hypothetical Model of HSP70B’ Regulation 171

List of Symbols

None

List of Conventions

Gene name with only first letter capitalized, i.e

Hsf1

Protein

Gene name entirely capitalized, i.e HSF1 Protein; often an abbreviation, complex or

compound name

xiv

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

Complex

xv

xvi

Real-time PCR